Method and apparatus for substantially isolating plant tissues

ABSTRACT

The present invention provides methods and devices for the rapid isolation of monocot plant embryos suitable for transformation or tissue culture. The invention includes mechanical devices for substantially isolating plant embryos for use as transformable explants. Media suitable for isolating plant embryos and methods for their preparation are also provided.

BACKGROUND OF THE INVENTION

This application is a Division of U.S. application Ser. No. 17/124,032,filed Dec. 16, 2020, which is a Division of U.S. application Ser. No.16/106,342, filed Aug. 21, 2018, now U.S. Pat. No. 10,918,032, which isa Division of U.S. application Ser. No. 14/339,934, filed Jul. 24, 2014,now U.S. Pat. No. 10,091,957, which is a Division of U.S. applicationSer. No. 13/035,908, filed Feb. 25, 2011, now U.S. Pat. No. 8,815,596,which is a Division of U.S. application Ser. No. 12/201,890, filed Aug.29, 2008, now U.S. Pat. No. 7,939,325 which claims the priority of U.S.Provisional Application Serial No. 60/969,287, filed Aug. 31, 2007,which are incorporated herein by reference in their entireties.

1. FIELD OF THE INVENTION

The present invention relates generally to substantially isolatingtarget plant tissues, such as embryos, which are suitable for genetictransformation or tissue culture.

2. DESCRIPTION OF RELATED ART

The preparation of tissues for plant propagation, regeneration andtransformation is time consuming and labor intensive, especially as itusually involves manual excision of transformable or culturable planttissues. For example, in corn (Zea mays), individual immature embryosare typically removed manually to provide genetically-transformableexplants. The manual excision of embryogenic tissues is laborious andrisks ergonomic injury to the worker. Moreover, when larger amounts oftransformable plant tissue are required for high-throughputtransformation and plant production, more workers must be employed andtrained to meet the increased demands. Additionally, there can besignificant variability in the quality of plant tissues obtained,depending on the skill level, care, attentiveness, and fatigue of theindividual workers.

The tissue variability and lack of amenability to automation in previoustechniques for isolating transformable plant tissues is problematic, aspoor quality tissues negatively impact subsequent tissue culture,genetic transformation, and plant propagation. Nonetheless, to produceeven a single transgenic plant suitable for commercial development anduse in agriculture, it may be necessary to produce tens of thousands ofindividual transformation events in a single species. Thus, there is agreat need in the art for improved methods of preparing target planttissues that are more efficient, reduce the overall ergonomic burden onworkers, reduce the amount of labor needed to process the plantmaterials, and/or that yield plant tissues that are of higher and moreconsistent quality than manually produced tissues.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for obtaining embryossuitable for tissue culture and/or genetic transformation, comprising atleast partially excising an embryo from a plant seed in a liquid mediumconsisting essentially of water and/or an osmotic agent with anosmolality of about 0 mOsm/kg to about 600 mOsm/kg, wherein the embryoremains viable for tissue culture and/or genetic transformationfollowing excision of the embryo from the plant seed. The osmotic agentmay further be an inert osmotic agent. In a further embodiment, themethod comprises at least partially excising a plurality of embryos froma population of plant seeds or ears in the liquid medium. The osmoticagent may be selected from the group consisting of mannitol, sorbitol,glucose, and sucrose, or other osmotic agents. In particularembodiments, the medium consists essentially of water and mannitol in aconcentration of from about 0.05 M to about 0.5 M, or sucrose in aconcentration of from about 0.05 M to about 0.5 M. The method mayfurther comprise the step(s) of genetically transforming the embryo, andregenerating a transgenic plant from the transformed embryo.

In certain embodiments of the method, the step of geneticallytransforming the embryo comprises use of a co-culture medium comprisinga bactericide. In particular embodiments the bactericide iscarbenicillin. In yet other embodiments, the co-culture medium comprisesabout 0.5-1.5 mg/L of 2,4-D.

The embryo may be from a member of the Poaceae, such as maize, rice,wheat, or millet. In particular embodiments, the embryo is a maizeembryo or a millet embryo. In other embodiments, the embryo may be asoybean embryo, a cotton embryo, or another dicot embryo.

In another embodiment, the method comprises preparing the liquid mediumin a media preparation system comprising: (a) an inlet for water; (b) aninlet for the osmotic agent; and (c) a chamber for mixing the water andosmotic agent to produce the liquid medium, wherein the inlet for waterand inlet for the osmotic agent are coupled to the chamber for mixing toallow delivery of the water and osmotic agent to the chamber for mixing.In certain embodiments, the inlet for water and/or the inlet for theosmotic agent are coupled to a chamber or chambers for holding the waterand/or osmotic agent. The method may further comprise coupling thechamber for mixing to a fluid jet apparatus. In other embodiments, themethod further comprises sterilizing the liquid medium with a sterilizerselected from the group consisting of a filter, a UV or gamma radiationsource, and a sterilizing heat source. The sterilizer may sterilize thewater and/or osmotic agent prior to entering the mixing chamber.Alternatively, the sterilizer sterilizes the liquid medium concurrentlywith and/or after entering the mixing chamber. In yet anotherembodiment, the method comprises sterilizing the liquid medium after theliquid medium leaves the mixing chamber.

The method may further comprise measuring the fill level of one or moreof the chamber(s) for holding water, the chamber for holding the osmoticagent, or the chamber for mixing the water and the osmotic agent. Infurther embodiments, the method comprises controlling delivery of thewater and the osmotic agent to the chamber for mixing the water and theosmotic agent. In particular embodiments, the method comprisescontrolling the delivery by electronically sensing the delivery of thewater and/or the osmotic agent.

In another aspect, the method for preparing a plant embryo suitable fortissue culture and/or genetic transformation comprises: (a) placingplant seed tissue comprising plant embryos and/or seed coats in anaqueous environment; (b) contacting the tissue with an agent thatselectively attaches to the embryos or seed coats; and (c) isolating atleast a first embryo based on the selective attachment of the agent tothe embryo or seed coat. In certain embodiments, the agent comprises gasbubbles. In particular embodiments, the gas bubbles have a largestaverage dimension of from about 100 microns to about 1 mm. In certainembodiments the gas bubbles may comprise a gas selected from the groupconsisting of air, oxygen, nitrogen, and a combination thereof. Further,in certain embodiments, step (c) comprises isolating the first embryobased on the buoyancy of the embryo.

The method may also comprise including in the aqueous environment asurfactant that reduces coalescence of bubbles with one another. Incertain embodiments, the surfactant is selected from the groupconsisting of a polyether, PPG (poly(propylene glycol)), and PEG(poly(ethylene glycol)). In particular embodiments, the PPG has amolecular weight of about 340 to about 3500 daltons, and the PEG has amolecular weight of about 100 daltons to about 9000 daltons.

In yet other embodiments, the agent comprises a second liquid that isimmiscible with the aqueous environment. The second liquid may beselected from the group consisting of vegetable oil such as canola oil,mineral oil, or other hydrophobic liquid compatible with survival andtransformation of the embryos.

In certain embodiments, the plant seed tissue comprises embryos producedby at least partially excising an embryo from a plant seed in a liquidmedium (aqueous environment) consisting essentially of water and/or anosmotic agent with an osmolality of about 0 mOsm/kg to about 600mOsm/kg, wherein the embryo remains viable for tissue culture and/orgenetic transformation following excising the embryo from the plantseed. In particular embodiments, the aqueous environment consistsessentially of a medium comprising water and/or an osmotic agent with anosmolality of about 7 mOsm/kg to about 500 mOsm/kg. The method maycomprise placing the plant seed tissue in the aqueous environmentwithout first separating embryo from non-embryo tissue. In a particularembodiment, the plant seed tissue is maize plant seed tissue. In otherembodiments, the plant seed tissue is soybean plant seed tissue orcotton plant seed tissue.

In another aspect, the invention provides an apparatus for preparingplant embryo tissue suitable for tissue culture and/or genetictransformation comprising (a) a container for holding plant seed tissuecomprising a plurality of plant embryos and non-embryo tissue, such asplant seed coats, in an aqueous environment; and (b) at least a firstnozzle for delivering to the aqueous environment an agent thatselectively attaches to the embryos or seed coats, wherein the nozzleproduces gas bubbles with an average diameter of from about 0.1 mm toabout 1 mm. In certain embodiments, the invention provides an apparatus,wherein the container is filled with media and plant seed tissuecomprising embryo and non-embryo tissue. In particular embodiments theapparatus further comprises a collector for separating embryo tissuebased on the buoyancy of the embryos within the aqueous environment. Inyet other embodiments, the gas bubbles may comprise a gas selected fromthe group consisting of air, oxygen, nitrogen, and a combinationthereof.

In yet another aspect, the invention provides a method for preparationof plant embryos suitable for tissue culture and/or genetictransformation comprising (a) directing a first stream of liquid mediumonto a corn kernel or other tissue comprising a plant embryo to extractendosperm from the kernel or tissue; and (b) directing a second streamof liquid medium onto the kernel or tissue to extract an embryo from thekernel or tissue. In certain embodiments, the liquid medium consistsessentially of water or an osmotic agent with an osmolality of about 7mOsm/kg to about 500 mOsm/kg. In a particular embodiment, the kernel maybe comprised on an ear of corn. In certain embodiments the method mayfurther comprise moving the ear of corn relative to the first and secondstream to remove the endosperm and embryo from the kernel in succession.In certain embodiments, the first and/or second stream comprises a widthless than the width of the corn kernel. In particular embodiments thefirst and/or second stream comprises a width of about 0.003″ and heightof about 1″, and the first and/or second stream is produced at apressure of from about 30 PSI to about 75 PSI.

In certain embodiments, the first and/or second stream may be directedfrom a nozzle that produces a laminar fluid flow stable at a distance ofat least 2.5″ from the nozzle. In yet other embodiments, the kernel maybe positioned about 1¾-2″ from the tip of the nozzle. In certainembodiments the first and/or second stream contacts each kernel in a rowof kernels found on the ear with substantially the same force.

In another aspect, the invention provides an apparatus for obtainingcorn or other plant embryos suitable for tissue culture and/or genetictransformation comprising (a) at least a first fluid jet for directing amedium onto a corn kernel or another tissue comprising a plant embryo;and (b) an apparatus for holding the kernel or other tissue in the pathof the medium. The corn kernel may be comprised on an ear of corn. Incertain embodiments, the apparatus for holding the kernel or othertissue comprises a sheet or a cylindrical sheet. In other embodimentsthe apparatus for holding the kernel or other tissue comprises a mesh ora plurality of slots; or a pressure cam or screw that applies force tothe tissue being held. Seed or fruit tissue may be held onto theapparatus for holding the kernel or other tissue by a mechanical force,friction, centrifugal force, or a suction force. In particularembodiments the holder may comprise a pressure cam, auger, or screw. Incertain embodiments, the apparatus for holding the kernel or othertissue is suspended in a gaseous phase, a liquid phase, or is partiallysuspended in gaseous and liquid phases. In other embodiments, theapparatus for holding the kernel or other tissue is fixed relative to afluid force, or is movable relative to a fluid force.

Another aspect of the invention comprises a method for preparation ofplant embryos suitable for tissue culture and/or genetic transformation,wherein the apparatus for holding the kernel or other tissue iscentrifuged in a container to apply force to the kernel, or othertissue.

The apparatus may be further defined as comprising a first and a secondfluid jet. Further, in certain embodiments the apparatus for holding thekernel comprises means for moving the ear of corn relative to the firstand second fluid streams to control the angle of contact between thefirst and second fluid streams and the kernel. In particular embodimentsthe apparatus further comprises a detector to identify excised endospermtissue and embryos.

In certain embodiments the apparatus further comprises at least a firstseparator to isolate embryos from non-embryo tissue. In particularembodiments, the separator separates embryos suitable for tissue culturefrom non-embryo tissue by a method selected from the group consisting ofsize exclusion, differential density and differential hydrophobicity.The apparatus may further be defined as comprising a sieve forseparating embryo from non-embryo tissue based on size. In a particularembodiment the apparatus for holding the kernel comprises at least afirst motor for moving the ear of corn relative to the fluid jet, orvice versa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one embodiment of an apparatus provided by the inventionthat uses positive mechanical pressure for substantially isolatingembryos, as described in Example 4.

FIG. 2 depicts one embodiment of an apparatus provided by the inventionthat uses fluid jet positive pressure to dislodge embryos from seeds asdescribed in Example 7. Legend: (A) robot with motion in X, Y and Zdimensions, (B) motor to rotate corn ear, (C) grasper, (D) handle tohold corn ear, (E), baffle to prevent material from splattering upwards,(F) flange to prevent material splattering upwards, (G) aperture forguiding fluid, (H) transparent tube, (I) corn ear, (J) shaking screen,(K) cheesecloth or other porous material, and (L) waste container.

FIG. 3 depicts one embodiment of a mounting mechanism using a magnetic“handle” by which a corn ear can be secured to a robot arm, as describedin Example 7.

FIG. 4 depicts one embodiment of a nozzle useful in certain aspects ofthe invention, as described in Example 7. This nozzle generates asubstantially uniform, flat sheet-like jet of fluid.

FIGS. 5A-5B depicts one embodiment of an apparatus useful in methods ofthe invention, as described in Example 13. This device includes a nozzlefor generating a substantially flat fluid jet and an optional suctionhead. FIG. 5A depicts a cross-sectional view of one embodiment of such adevice, showing how the nozzle, optional suction head, and corn ear maybe positioned relative to each other. FIG. 5B schematically depicts acorn ear positioned in the device. Legend: (A) base, (B) nozzle, C)shaft attached to base of ear, (D) suction head, (E) corn ear, and (F)aperture for guiding fluid flow.

FIG. 6 depicts one embodiment of a component useful for applyingnegative pressure useful in methods of the invention, as described indetail in Example 13. Legend: (A) one or more apertures for guidingfluid flow.

FIGS. 7A-7C depict different views of an embodiment of a device thatuses a combination of forces and is useful in certain aspects of theinvention, as described in detail in Example 13. This device includes ahead with a leading edge capable of applying a predefined amount ofmechanical pressure to the base of kernels that previously have had thepericarp opened or truncated and a component for applying negative fluidpressure. This device can further include a means for dispensing fluidor for guiding fluid flow.

FIG. 8 depicts an embodiment of the invention in which the top of acylinder is wholly, partially or substantially covered with a membraneor a sheet of soft material that is slightly smaller than the diameterof the corn ear and the handle to which the corn ear is attached.Specifically, a silicone rubber membrane splash guard ( 1/32″ thick)from McMaster-Carr (McMaster-Carr, Atlanta, Ga.; e.g. cat. #9010K11) isshown. A 1″ diameter hole was made in the membrane. The corn ear wasable to move both down through the opening and also back up through it.

FIG. 9 depicts a 1 liter polymethylpentene graduated cylinder with thebottom cut off. Three fluid jets are threaded into holes cut in the wallof the cylinder. The entire apparatus can be autoclaved prior to use.For operation, the top of the cylinder can be fitted with a splash guardand the corn ear lowered into the cylinder so that the fluid jetsdislodge the contents of each kernel. The dislodged material then fallsout the bottom of the cylinder for further processing.

FIG. 10 depicts a corn cob showing the location of the embryo within anindividual kernel. Also shown is one embodiment of the invention inwhich a liquid jet is directed to the basipetal side of the kernel,opposite the acropetal side where the embryo is located.

FIG. 11 illustrates an embodiment of a Media Preparation System providedby the invention.

FIG. 12 illustrates a general view of the mixing chamber of the MediaPreparation System provided by the invention.

FIG. 13 illustrates a close up view of the mixing chamber, lower end,with positioning plate and quick connect mounting.

FIG. 14 depicts a close up of the lower end of the mixing chamber, withpositioning device and supporting plate.

FIG. 15 depicts a close up of the lower portion of the Media PreparationSystem in work position.

FIG. 16 depicts a close up of the upper portion of the Media PreparationSystem in work position.

FIG. 17 comprises a cross sectional view of an apparatus for phasedseparation of corn embryos.

FIG. 18 illustrates a spiral shaped limewood bubble dispersion device.

FIG. 19 illustrates embryos floating on top of froth produced by aspiral shaped limewood bubble dispersion device.

FIG. 20 illustrates separation of embryos from endosperms by use of afloatation process.

FIG. 21 depicts a vacuum filter device used to harvest embryos from thefroth at the surface of an air-fluid interface of an embryo separator.

FIG. 22 comprises a schematic diagram of an embryo separation device asdescribed in Example 19.

FIG. 23 depicts an alternative apparatus for separating embryos byflotation as described in Example 20.

FIG. 24 illustrates a top view of an embryo extractor in a combinationdevice for extracting and separating corn embryo tissue for tissueculture.

FIG. 25 depicts an end view of an extractor for rotating and extractingembryos from multiple ears.

FIG. 26 illustrates a side view of an embryo separator.

FIG. 27 illustrates an exemplary liquid level regulator.

FIG. 28 illustrates exemplary mesh and slot holders for seed and/orfruit tissue; (FIG. 28A; mesh holder; FIG. 28B; slotted holder).

FIG. 29 depicts a sheet and a cylindrical embodiment of a seed or fruitholder.

FIG. 30 illustrates an embodiment of a seed or fruit holder comprising apressure cam or screw.

FIG. 31 illustrates an explant separator comprising a limewood bubbledispersion device utilized for separation of cotton embryogenic tissue.

FIG. 32 depicts cotton explants floating atop the froth produced in themicrobubble dispersion device shown in FIG. 31 .

FIG. 33 shows a comparison of the purity of cotton explants producedeither via excision and sieving (left) or excision, sieving, and thenadditionally purified using microbubble technology (right).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, when taken in context of thepresent specification. Where there are inconsistencies between the textof the specification and the material incorporated by reference, thedefinitions and meanings provided in the present specification areintended. The nomenclature used herein and the manufacture or laboratoryprocedures described below are well known and commonly employed by thoseof skill in the art.

The phrases “substantially isolated” or “extracted” refer to theprocessing of a target tissue (e.g., an embryo or other tissue explant)that resides in or forms part of a larger tissue complex (e.g., a seed)such that the target tissue is physically separated from at least halfof the larger complex. In some embodiments, a substantially isolatedtarget tissue may be physically separated from at least about 20%, 30%,40%, 50%, 60%, 70%, 85%, 90%, 95%, 97%, 98%r 99% of the larger complex,or any fraction thereof. In other embodiments, the target tissue isphysically separated from more than about 80% to about 100%, about 90%to about 100%, or about 99% to about 100% of the larger complex, or anyfraction in between. In some embodiments, the target tissue may bephysically separated from about 100% of the larger complex.

While a substantially isolated target tissue is physically separatedfrom some percentage of the larger complex, it does not necessarily haveto be purified from that complex. In other words, the substantiallyisolated target tissue may remain in a batch with the larger tissuecomplex, so long as the target tissue is physically separated from thecomplex (as described above). In some embodiments, however, it may bedesirable to remove some, or all, of the separated complex from thesubstantially isolated target tissue. All such embodiments are withinthe scope of the present invention.

The phrase “target plant tissue” refers to a portion of a plant tissueor seed that one seeks to substantially isolate. In the presentinvention, target plant tissue refers to any portions of a plant orplant seed that can be substantially isolated and used for genetictransformation or tissue culture. In some embodiments, the target planttissue is an embryo, such as an immature embryo from a monocot such ascorn. In other embodiments, the target plant tissue is from adicotyledonous plant such as soybean (Glycine sp. including Glycine max)or cotton (Gossypium sp. including G. hirsutum).

The phrase “suitable for genetic transformation” and “suitable fortissue culture” refer to plant tissues that are competent fortransformation or growth in under suitable plant culture conditions,respectively. One of skill in the art can readily determine if aparticular target tissue is suitable for genetic transformation ortissue culture by using routine experimentation. For example, a samplefrom a batch of substantially isolated target tissues may be cultured onsuitable plant media (also known to those of skill in the art) todetermine if the tissues are capable of growth and regeneration.Similarly, samples of substantially isolated target tissues can besubject to transformation and screened for the presence of aheterologous nucleic acid molecule. Such techniques are routine and canrapidly identify which tissues are competent for transformation ortissue culture and which, if any, are not.

Substantially Isolating Target Plant Tissues

The present invention provides methods of substantially isolating targetplant tissues suitable for genetic transformation and/or tissue culture.In some embodiments, the target plant tissue is an embryo. In oneembodiment, the embryos are monocot embryos, such as from maize. In someembodiments, the substantially isolated target tissue may be isolated inwhole or in part. For example, a batch of substantially isolatedimmature embryos may include intact embryos, partial embryos, ormixtures thereof. Preferably, the intact and/or partial tissues aresuitable for genetic transformation, tissue propagation, plantregeneration and other tissue culture applications.

As tissues are being isolated using, for example, by streams of aselected media, a collection receptacle may be provided. In someembodiments, it is useful to provide a covering for such collectionreceptacles, in order to improve the efficiency of the apparatus, reducemess, prevent undesirable splashing from the jet stream, and/or limitany escape of extracted tissues during harvesting. Any suitablereceptacle or receptacle covering may be employed. Examples are givenelsewhere in this application and are also known to those of skill inthe art.

Suitable coverings for the receptacle may include those made of metals,wood, glass, meshes, fabrics, plastics, rubbers, latex, acrylics, andfunctionally equivalent materials. In some embodiments, the material isflexible so as to allow penetration and removal of an ear of corn whilemaintaining a substantially water tight seal around the ear during theextraction process. The material may be provided with a suitable openingto allow entry and removal of an ear of corn. In some embodiments, thematerial is solid and contains a flexible hole for receiving and holdingthe ear during extraction. In other embodiments, the material isflexible. Such flexible materials may be stretched over the receptacleto form a liquid tight fit, but allows for insertion of an ear either bypenetrating the material or by providing an opening to receive the earof corn. In still other embodiments, the material is a mesh or screenthat has a flexible opening.

The coverings may be removable or semi-permanently attached. In someembodiments, the materials are held by an elastic band or equivalentsecuring means. In other embodiments, the covering is held in place byweights, friction collars, hooks, snaps, or other functionallyequivalent securing means.

The covering may be made of a flexible material and have varyingthickness. These factors may be varied in order to achieve the desiredeffects for insertion and extraction of ears of corn. The followingtable (Table 1) illustrates some hardness and thickness parameters forsilicone coverings. The invention, however, is in no way limited tothese few choices.

TABLE 1 Hardness and thickness parameters of coverings. Durometerhardness Membrane thickness (in.) 10A 1/32 10A 1/16 20A 1/32 20A 1/1640A 1/32 40A 1/16

In one embodiment, the collection receptacle is covered with a membraneor sheet of soft material that is slightly smaller than the diameter ofthe corn ear and handle to which it is attached. In an alternativeembodiment, small incisions are made in the membrane to provide moreflexibility. The membrane may be attached to the cylinder by a meanssuch as an elastic band or a friction fit collar, or the like. FIG. 8illustrates an embodiment of the covering and attachment means describedabove.

In some embodiments, the material is autoclavable. Autoclavablematerials are well known to those of skill in the art. For example asoft material such as a soft silicone rubber sheet may be used. One ofskill in the art is aware of the possible materials and physicalarrangements that will permit extraction while providing a collectionvessel that reduces escape of extracted tissue and prevents undesirablesplashing.

Suitable procedures for plant tissue culture and regeneration are wellknown in the art. See, for example, U.S. Pat. No. 5,550,318 to Adams etal., U.S. Pat. No. 5,780,708 to Lundquist et al., United States PatentApplication Publication Number 2004/0210958 to Duncan et al., UnitedStates Patent Application Publication Number 2004/0016030 to Lowe etal., and United States Patent Application Publication Number2004/0244075 to Cai et al., which disclose transformation methods usefulwith corn, and United States Patent Application Publication Number2003/0024014 to Cheng et al., which disclose transformation methodsuseful with wheat, all of which are incorporated by reference in theirentirety herein. The tissue culture applications can include at leastone process selected from transformation, callus formation, formation ofdifferentiated plant tissue, formation of at least one mature plant,formation of at least one fertile mature plant, and combinations ofthese processes. The plants regenerated from the extracted immatureembryos may be regenerated, for example, through differentiation ofde-differentiated tissue (calli). Regenerated plants can be grown tomaturity to provide mature plants, including fertile mature plants. Theextracted immature embryos and extracted non-embryo tissues may also beused for other purposes, such as, but not limited to, genetic orbiochemical analysis.

The methods and apparatuses of the present invention can be applied toany monocot plants of interest. Preferred monocots include, but are notlimited to, members of the family Poaceae, including grain crops such ascorn (maize), wheat, barley, oats, rye, sorghum, millet, and rice.Particularly preferred monocots include Zea species, including corn (Zeamays), and millets (e.g. Pennisetum glaucum, Pennisetum sp., Setariasp., Panicum sp.) which have multiple kernels (seeds) typically held inrows on an ear.

In general, the monocot seeds from which the target tissues aresubstantially isolated are provided in any suitable manner. For example,seeds may be attached to the ear or head on which the seeds grow; insome embodiments the monocot seeds may be removed from the ear or headprior to substantially purifying the target tissue.

In some embodiments, an opening in the pericarp or seed coat of themonocot seeds is provided. This may be accomplished by any suitabletechnique, such as, but not limited to, making a hole, puncture, orincision with a needle, awl, blade, or other suitable implement. In someapplications of the method, no pericarp tissue need be removed; in otherembodiments, the opening of the pericarp may include removal of at leastpart of the pericarp and possibly of some non-embryo tissue (e.g.,endosperm). Preferably, the opening is sufficient to substantiallyseparate the embryo from the seed. In some embodiments it may benecessary only to weaken the pericarp sufficiently (for example, byabrasion, or by other physical, chemical, or enzymatic treatment) sothat application of force to the seed results to substantial isolationof the target tissue, such as the embryo.

In such a method force is generally applied to seeds sufficient tosubstantially isolate the target tissue, such as an immature embryo,wherein the substantially isolated target tissue is suitable for genetictransformation and tissue culture. Force may be applied to multipleseeds consecutively or simultaneously. The applied force can becontinuous or non-continuous (for example, pulsed or wave-like force),and is generally mechanically applied, that is, through use of a deviceor machine rather by human hand. The amount of force applied ispreferably sufficient to overcome the adhesion of the target (e.g.,embryo) and non-target (e.g., non-embryo tissue such as endosperm) fromeach other, thus allowing separation of the target and non-targettissues. Any suitable force or forces may be employed for removal of thetarget tissue from its seed, and multiple forces may be used incombination, sequentially or simultaneously. Suitable forces include,but are not limited to, fluid jet positive pressure, liquid jet positivepressure, mechanical positive pressure, negative pressure, centrifugalforce, linear acceleration, linear deceleration, fluid shear, fluidturbulent flow, suction, and fluid laminar flow. Fluid forces can beexerted by any fluid, e.g. gases or liquids, or combinations of both.

Since a corn embryo is located on the acropetal side of a kernel, it ispossible to direct a liquid jet to the basipetal side of the kernel, ifdesired, to successfully eject the embryo (See, e.g., FIG. 10 ). In suchan arrangement, the full force of the jet is generally not directlyimpacting the embryo. Rather, the substantial amount of the force isonly indirectly applied to the embryo itself. Thus, stronger forces maybe applied in the apparatus to accelerate the removal of embryos withoutsubstantially increasing the damage to the embryos being removed.

Higher impact forces can be provided by forcing higher quantities ofliquid through the apparatus of the present invention. In someembodiments, however, higher impact forces can be generated withoutusing more liquid. For example, in some embodiments, the size of the jetopening is reduced so that the same volume of liquid can be used at ahigher velocity. As the energy of a moving object is proportional to thesquare of the velocity, a jet with the same volume can have much greaterenergy. A simple equation for kinetic energy of a moving object is equalto (½)(m)(v²). The calculation of the actual impact energy of a liquidjet would also take into account other factors known to those of skillin such art. Additionally, some embodiments may use a combination ofincreased fluid and changes in the size of the jet openings to achievethe desired force or energy.

Nozzles with gpm ratings of about 0.01 to about 0.25, for example, maybe used in the present invention, or about 0.01 to about 0.2, or about0.01 to about 0.1, or about 0.01 to about 0.09, 0.08, 0.07, 0.06, 0.05,0.04, 0.03, 0.02, or 0.015, or any whole number or fraction in betweenthese amounts. In some embodiments, using nozzles with low gpm ratings,like 0.033 or 0.021 gpm may be used. When such nozzles are used withhigher pressure but directed at the opposite side of the kernel from theembryo, accelerated embryo harvesting may be achieved while avoidinginjury to the embryo.

In some embodiments, multiple jets are provided in an apparatus of thepresent invention. Such an apparatus is useful to equalize the forceexerted by the fluid jets while decreasing the time needed to harvestthe embryos from an ear of corn.

In some embodiments, the apparatus may have 2, 3, 4, 6, 8, 10 or morejet openings/nozzles as desired for conveying fluid force. In oneembodiment, there are three openings. Such a device is depicted in FIG.9 . In some embodiments, the openings are provided as narrow-angle flatstream jets, oriented horizontally. However, other embodiments of thejet openings are provided elsewhere throughout this application. Thestainless steel spray nozzle components connecting directly into thepolymethylpentene graduated cylinder depicted in FIG. 9 are shown inTable 2:

TABLE 2 Spray nozzle components. Spraying Systems Component descriptionCat. No. Comments Wall mount adapter 4865 SS Threads into cylinder wallSpray tip, solid stream TP000067-SS 0° spray angle, 0.067 gal/min (~253ml/min) Screen strainer (200 mesh) 6051-SS-200 Removes debris that canclog spray tip Female body CP1321-SS Provides connection to ¼″ male NPTpipe threadThe remaining components are mostly tubing and fittings with a malequick disconnect fitting at the fluid source to the right of the photo(FIG. 9 ).

A method and apparatus of the invention can further provide forseparating substantially isolated target tissue, such as immatureembryos, from associated non-embryo tissue such as endosperm, glumes,and seed coat or pericarp tissues. Separation may be accomplished by oneor more suitable techniques, including, but not limited to, separationby size exclusion (for example, by filtration in one or more filteringsteps), separation based on hydrophobicity, hydrophilicity, or otherforces, and separation by mass or density differentials (for example,separation by centrifugation, settling, and decanting). The separationstep or steps can be optional, for example, where no additionalisolation of intact or partial embryos is necessary for their use intissue culture.

The invention is particularly suitable to applications where a largenumber of target tissues must be provided, for example, inhigh-throughput processes or screening, or in batch processing forgenetic transformation or tissue culture. Automation of the method ispossible, for example, by employing robotic or mechanical handling ofthe corn ears or seeds, opening of the pericarp, application of force tothe seed, or the optional separation steps. Such automation may useoptical or mechanical sensors to aid in positioning the corn ears orseeds relative to the applied force or forces, or in the separationsteps. In one embodiment, the method provides substantially isolatedembryos at a rate of between about 250 to 50,000 or more embryos perday, or between about 500 to about 100,000, or about 250 to about50,000, or about 250 to about 20,000, or about 250 to about 10,000, orabout 250 to about 5000, or about 250 to about 3000, or about 250 toabout 1000 embryos per day; or between about 1,000 to about 50,000embryos per day, and the like, including any fraction or whole number inbetween any of the aforementioned ranges. As noted above, the presentinvention overcomes the significant output limitations of manualexcision of embryos.

Apparatus for Substantially Isolating Target Plant Tissues

The present invention also provides an apparatus for substantiallyisolating target tissues, such as corn embryos, that are suitable forgenetic transformation or tissue culture. In one embodiment forseparating corn embryos, such an apparatus may comprise at least oneaperture for guiding a fluid stream, wherein the fluid stream contactskernels on the corn ear and substantially isolates embryos from thekernels. Generally, it is preferred that the fluid stream contact asmany of the kernels in a given period of time as is convenient, so as tomore rapidly isolate embryos. The aperture can include a single apertureor multiple apertures, for example, single or multiple nozzles, whichcan include nozzles which produce flat, round, oval, fan-shaped or otherpatterned jets of fluid, and adjustable, moving, or stationary nozzles,and can generate a fluid flow of any suitable type and medium. Fluidsmay be gases, such as air, nitrogen, or gas mixtures, liquids, such aswater, physiological saline, or various culture media, media asdescribed below, or combinations. Suitable fluid flows include, but arenot limited to, fluid jets, such as single or multiple columnar jets;flat, cone-shaped, or fan-shaped jets or sprays; and sheet-like jets,laminar fluid flow, and turbulent fluid flow. Suitable fluid flows canresult in a variety of forces to remove the embryo from its kernel,including positive pressure or negative pressure or both; such forcescan be uniform or non-uniform, continuous or non-continuous (such as apulsed or wave-like force), or in any combination thereof.

An apparatus of the invention may further include a means for moving thetarget tissue being substantially purified and the fluid stream,relative to each other. For example, either the ear of corn containingseeds or the fluid stream, or both, may be moved. Various embodiments ofthe apparatus can be used with single or multiple, intact or partialears of corn. For example, the corn ear or ears can be secured to aholder or grasper, which is moved relative to the fluid stream. In otherembodiments, however, the corn ear or ears need not be individuallysecured to a holder but can be freely movable so as to allow multiplekernels to be contacted by the force used to remove the embryos from thekernels. The means for moving at least one corn ear relative to thefluid stream can rotate the corn ear and the aperture relative to eachother, or can move the fluid stream along the longitudinal axis of thecorn ear, or can provide any suitable three-dimensional movement of thecorn ear and the aperture relative to each other, such as a combinationof rotation and longitudinal motion.

The invention further provides a separator for separating target tissuesfrom non-target tissues. For example, embryos may be separated fromnon-embryo tissues, wherein the separated embryos comprise at least somecorn embryos suitable for genetic transformation or tissue culture.Separators can work by a mechanism including, but not limited to,separation by size exclusion (for example, using a mesh, screen,perforated surface, or other device capable of excluding objects of acertain size), separation based on hydrophobicity or other attractiveforces (for example, using a material, solid or fluid, that can attractor repel the embryos), and separation by mass or density differentials(for example, using a centrifuge, or using solutions for differentialsettling). In certain embodiments, the separator can be optional, forexample, where no additional isolation of intact or partial embryos isnecessary for their use in genetic transformation or tissue culture.

The substantially isolated immature embryos include at least someembryos, such as immature intact or partial embryos, suitable for tissueculture applications, transformation, callus formation, directembryogenesis, formation of differentiated plant tissue, formation of atleast one mature plant, formation of at least one fertile mature plant,and combinations of these processes, as described above. Thesubstantially isolated immature embryos and non-embryo tissues may alsobe used for other purposes, such as, but not limited to, genetic orbiochemical analysis.

The present invention further provides an apparatus for substantiallyisolating in a mechanical process multiple corn embryos suitable forgenetic transformation or tissue culture from at least one immature cornear. In one embodiment the device comprises at least one componentselected from (a) at least one solid surface for applying mechanicalpositive pressure to the exterior of kernels on an immature corn ear;(b) an aperture for guiding a fluid flow, wherein the fluid flowcontacts kernels on the corn ear; and (c) an aperture for applyingnegative fluid pressure that contacts kernels on the ear; wherein thecomponent applies force sufficient to substantially isolate embryos fromthe kernels suitable for genetic transformation or tissue culture. Theforces may be applied to multiple seeds consecutively or simultaneously,in a continuous or non-continuous manner, and are generally appliedmechanically and not manually. Multiple forces may be used incombination, sequentially, or simultaneously. Suitable forces include,but are not limited to, fluid jet positive pressure, liquid jet positivepressure, mechanical positive pressure, negative pressure, centrifugalforce, linear acceleration, linear deceleration, fluid shear, fluidturbulent flow, and fluid laminar flow. Fluid forces can be exerted byany fluid, gases or liquids or combinations of both.

Combination apparatuses of the invention can optionally include a meansfor moving the at least one corn ear relative to the source or sourcesof force for isolating embryos or parts thereof. The ear or ears may bemoved relative to the source of force so that the force or forcescontact as many of the kernels in a given period of time as isconvenient, so as to more rapidly isolate embryos.

Combination apparatuses of the invention can further include at leastone means for further separation of the substantially isolated immatureembryos suitable for genetic transformation or tissue culture.Separators may function by a mechanism, including, but not limited to,separation by size exclusion, separation based on attractive forces, andseparation by mass or density differentials.

Liquid Media for Excising Corn or other Plant Embryos

The present invention provides in one embodiment media and methods forsubstantially isolating target plant tissues suitable for genetictransformation or tissue culture. For example, a fluid jet apparatusutilizes liquid for excising embryos, requiring substantial quantitiesof liquid for excision. Thus, for instance, about 20 L of a liquidmedium may be needed for excising embryos from one ear of corn. Themedium is therefore preferably easily prepared (consisting essentiallyof one or two ingredients), sterilizable, for instance through anin-line filtration unit, and can flow through a fluid jet apparatusoperating at a pressure of about 30-75 psi, for instance about 40-60psi. Further, it preferably does not require pH adjustment prior to use.To save resources and expense, it is also, preferably, reusable.

Culture media such as Lynx #1013 inoculation medium and Lynx #1902(half-strength Lynx #1013) have been successfully used in an automatedmethod and apparatus for excising corn embryos for use in tissueculture. However, these media have multiple ingredients, and require pHadjustment prior to use. Thus, the current inventors have developedmedia that can be used to optimize efficiency of an automated embryoexcision method for tissue culture. Surprisingly, the inventors found inparticular that a media consisting essentially of water and/or anosmotic agent with an osmolality of about 0 mOsm/kg to about 600 mOsm/kgto be useful.

In certain embodiments, an osmolality of about 0 mOsm/kg to about 600mOsm/kg for the osmotic agent is preferred, including about 7 mOsm/kg toabout 500 mOsm/kg, about 13 mOsm/kg to about 300 mOsm/kg, about 25mOsm/kg to about 300 mOsm/kg, about 50 mOsm/kg to about 300 mOsm/kg,about 13 mOsm/kg to about 200 mOsm/kg, about 13 mOsm/kg to about 100mOsm/kg, or about 100 mOsm/kg to about 300 mOsm/kg. In specificembodiments, a media provided consists essentially of sterile distilledwater, dilute calcium chloride (e.g. about 10 ppm), 0.05% MES, pH5.4-5.8, about 5% sucrose +/−MS salts, or about 0.05-0.5 M mannitol,including 0.1-0.2 M mannitol solutions. Although the transformationfrequency (TF) of plant embryos excised by use of some such liquid mediamay be somewhat reduced from that found when using, for instance, theLynx #1013 Inoculation Medium, the significant time and costefficiencies arising from use of simpler media can outweigh anyreduction in TF by allowing production of more explants using the sameamount of resources.

In specific embodiments of the invention, an excision medium comprisesas an osmotic agent mannitol and/sucrose, such as media consistingessentially of 0.05-0.5M mannitol. Such media was found capable ofproducing viable explants. In particular embodiments, an excision mediumconsists essentially of about 0.1-0.2 M mannitol. The osmolality of a0.2 M solution of mannitol in water is, for instance, about 225 mOsm/kg.In other embodiments, sterile distilled water, as well as 5% sucrose(w/v) were also found to be simple, yet effective, media for use withthe invention.

Media Preparation System

A Media Preparation System (MPS) for use with a fluid jet apparatus isanother embodiment of this invention. The MPS, for instance, may be usedto supply an excision medium described above. The MPS may consist of ahousing (MPSH) and a mixing chamber (MC). The MPS may be made from asuitable material, such as aluminum (e.g. 7075 aircraft grade aluminum)or steel. The mixing chamber generally comprises a tank with an upperand a lower end, and may comprise a flange and a cover plate. The coverplate was generally provided with one or more o-ring(s) or other sealingmeans which seal the mixing chamber. The cover plate comprises openingsfor inserting and removing liquid and other media ingredients. Liquidand other media ingredients may be added via one or more inlets,comprised in the mixing chamber, which may communicate with a chamberand/or supply line that holds and/or delivers a supply of theingredients. The cover plate may also comprise an electronic sensor forsensing a specified volume of medium that is being prepared. The mixingchamber may be oriented on a supporting plate and held in a workingposition by holding means, such as mounting pins and positioningbrackets. The supporting plate may be slidably connected to the MPSH. Incertain embodiments, the mixing chamber is connectable to a fluid jetapparatus, for instance via steel or polycarbonate tubing.

A mixer assembly may be attached to the cover plate or other part of themixing chamber. The assembly may comprise an impeller and shaft mountedon a means for suspension such as a high temperature ball bearing. Themixer may be autoclaved prior to use. The MPS may further comprise aProgrammable Logic Controller (PLC) in communication with sensors tomonitor ingress of components, media preparation, and subsequent removalof media, for instance via a fluid outflow tube to the fluid jetapparatus. One or more fluid inflow and outflow tubes may comprise aninline filtration unit to sterilize the prepared media.

Apparatus for Phased Excision of Embryos

Methods and apparatuses for preparing multiple embryos suitable fortissue culture are embodiments of the invention, wherein the methodscomprise use of at least two fluid streams for substantially extractingan embryo and endosperm from a seed kernel, and the apparatuses compriseat least one aperture for guiding a first fluid stream and a secondfluid stream, for substantially extracting an embryo from a seed kernel.The apparatus may further comprise means for moving at least one seedear relative to the first and second fluid streams. In certainembodiments, the seed ear or seed kernel is a corn ear or corn kernel.

The fluid streams may be gas streams or liquid streams. In certainembodiments, the fluid streams comprise liquid, and in particularembodiments the liquid consists essentially of distilled water, about 5%sucrose, or about 0.05 M-0.5 M mannitol, for instance about 0.1-0.2 Mmannitol. The fluid stream may exert a force for substantiallyextracting an embryo, comprising one or more forces selected from thegroup consisting of fluid jet positive pressure, liquid jet positivepressure, mechanical positive pressure, negative pressure, centrifugalforce, linear acceleration, linear deceleration, fluid shear, fluidturbulent flow, and fluid laminar flow. The fluid stream may becontinuous or pulsed. In particular embodiments the fluid stream issterile.

An apparatus of the invention may further comprise a means for detectingexcised endosperm and embryo tissue, and a means for channelingendosperms or embryos for automation. The means for detecting and themeans for channeling may be linked, for instance electronically, toassist in automation. The apparatus may also comprise at least oneseparator for separating embryos from non-embryo tissue, wherein theseparated embryos are embryos suitable for tissue culture. The separatormay separate embryos from non-embryo tissues by differentialhydrophobicity, by size-exclusion, or by density differential. Theembryo tissue may be cultured, transformed, and/or regenerated to yieldat least one fertile plant. In particular embodiments, the embryo is acorn embryo, and the plant is a corn plant.

Separation of Embryos by Flotation

The invention further provides an apparatus and methods for separationof embryos based on differential affinity of a selected agent relativeto non-embryo seed tissue. In certain embodiments, embryos, such as cornembryos produced by a fluid jet excision process, are contacted by, andattach to, bubbles via hydrophobic interactions.

In other embodiments, seed coats are contacted by, and attach to,bubbles. The bubbles may comprise one or more gases selected from thegroup consisting of an inorganic gas such as argon, air, O₂, N₂; and acovalent organic gas such as methane, ethane, or propane, that do notseriously affect the viability and transformability of the excisedembryos.

Bubble size was found to play an important role in the efficiency of theseparation process. In practice, (gas) bubbles displaying a range ofsizes may be generated. However, any bubble size that is suitable forattaching to the embryos or seed coats and raising them to the surfaceof the fluid is within the scope of this invention.

The size distribution of bubbles may vary with a number of factors suchas:

Frit uniformity: The openings between particles composing a frit may bevariable, allowing various size bubbles to form.

Gas flow rate: The flow rate of gas through the bubble-generating fritalso affects bubble size—e.g. fast gas flow results in larger bubbles.

Surfactants and their concentrations: Media additives such as mannitoland PEG as well as proteins and other substances with surfactantproperties released from the disrupted corn kernels may affect bubblesize.

Bubble merging: As bubbles rise toward the top of the tank some of themmerge with other bubbles.

Hydrostatic pressure: Bubbles near the bottom of a flotation tank willon average be slightly smaller because of the higher pressure at thebottom of the tank, although this effect would be minor in view of thesize of the flotation tank.

Further, it is not necessary for a single bubble to carry a singleembryo or seed coat all the way to the surface. Instead, an embryo orseed coat may be only partially carried to the surface by one bubbleonly to be carried further by sequential attachment of other bubbles.Also, an embryo or seed coat may not reach the surface and becomeembedded in the froth the first time around, but may circulate a numberof times before arriving at the interface of the air and liquid phases(e.g. the “froth”).

Large bubbles, for example, about 1 mm in diameter (i.e. if spherical;or largest dimension if non-spherical), or greater, do not achieve goodseparation for several reasons. For instance, such bubbles move fastenough that they hydrodynamically push embryos as well as debris out oftheir way. Thus such large bubbles tend to not adequately contact theembryos. Such bubbles also do not have sufficient contact time with theembryos or seed coats to allow efficient bubble attachment. Finally,such bubbles generate a high enough shear force while rising to thefluid surface that any embryos that are adhering to the bubbles tend tobe detached before reaching the surface.

Small bubbles, e.g., bubbles around 100 micron diameter, or less, attachto debris as well as embryos. This is because the bubbles move slowlyenough through the fluid that they do not push smaller material (e.g.endosperm fragments) out of the way. Thus, such bubbles contact with andattach to both embryos as well as other smaller debris. Finally, suchbubbles do not generate enough shear force to efficiently dislodgeattached debris.

Mid-sized bubbles (between 100 μm and 1 mm in size) were comparativelymore effective in achieving embryo purification. Such bubbles apparentlyproduce a low enough hydrodynamic displacement to allow bubble contactwith embryos, thus allowing attachment to occur. Additionally, suchbubbles do not generate enough shear force to dislodge attached embryos.

Another aspect of the separation process relates to the physical natureof the gas present in the bubbles, i.e., the selectivity of the gasbubbles for the embryos or seed coats. Air is approximately 21% oxygenand 78% nitrogen. Oxygen and nitrogen are both covalently bondeddiatomic molecules and have a strong affinity for the similarly covalent(nonpolar) waxy portions of the embryos, such as cotton embryos. Incontrast, such gases are less efficient in attaching to the relativelypolar endosperm debris, which remain floating in the medium. In short,the bubbles compete with the aqueous fluid (e.g. water ormannitol/water) to attach to the waxy surface of the embryos. Since bothoxygen and nitrogen molecules exceed water molecules in their covalentcharacter, they attach preferentially to the embryo surface. Thus, therelatively lipophilic nature of oxygen and nitrogen and the relativelyhydrophilic nature of the aqueous medium explains the binding of bubblesto lipophilic portions of, for instance, the cotton embryo surface.Alternatively, bubbles were found to efficiently attach to soybean seedcoats which rose to the “froth” at the liquid-air interface, whilesoybean embryos, including soybean embryonic axes and cotyledons, werefound to remain in the liquid phase. Thus, agents that differentiallyattach to various seed components and direct them to distinct locations,phases, or fractions may be utilized to enrich a given location, phase,or fraction for a seed component, such as an embryo or a portion of anembryo.

In one embodiment, air is used for preparing bubbles. Other useful gasesin practicing this invention include O₂ or N₂, as well as covalentinorganic gases other than O₂ or N₂ including noble gases, such asargon. Covalent organic gases such as methane, ethane, and propane andother lipophilic gases or gas mixtures that do not seriously affect theviability and transformability of the corn embryos may also be used.

In the broadest sense, the “bubbles” may be made of any material thatdisplays preferential binding to the embryos, including solids orliquids or mixtures thereof that selectively attach to embryo surfaces.Shapes such as a moving plane surface, e.g., a belt or disk to whichembryos would selectively attach may be utilized. In one embodiment,embryos floating on the surface of liquid medium were preferentiallyattached to a sheet of hydrophobic filter paper (Whatman PS waterrepellent phase separating paper, impregnated with silicone). In anotherembodiment, canola oil mixed with a suspension of embryo and debrispreferentially attached to the embryos, and carried them to the surfaceas the oil rose to the surface.

Bubbles coming into contact with each other can merge in milliseconds.Stabilizing the bubbles as they rise preserves an appropriate bubblesize distribution as well as promotes a stable froth at the top of theflotation liquid. Several surfactants were shown to be useful inpreventing coalescence and stabilizing bubbles. Preferably, thesurfactant is an ionic or non-ionic surfactant, such as a polyether. Thepolyether may be PEG. The PEG can have an average molecular weight ofabout 100, 300, 400, 600, 900, 1000, 1450, 3350, 4500, 8000, and 9000.Preferably, the PEG has a molecular weight of about 8000. The PEG isused at a water soluble concentration sufficient to prevent prematurecoalescing of bubbles. Another suitable polyether is PPG. The PPG has amolecular weight of about 340 to about 3500. Preferably, the PPG has amolecular weight of 340. The PPG can be used at a water solubleconcentration sufficient to prevent coalescing of bubbles.

In one embodiment, bubbles may be produced by a fitted fine pore glassdispersion tube (Chemglass, NJ, USA) connected to an aquarium pump forpumping air through a sterile filter and into the glass dispersion tubefor creating bubbles. The dispersion tube can be placed into a containersuch as a graduated cylinder or a beaker containing a mixture of embryosand debris in a suspension. However, the strong convection currentscreated by the large population of bubbles generated by this deviceproduced enough shear force to reduce its effectiveness in separatingembryos from debris. Alternatively, the bubbles may be produced byanother method, such as a multiple point source bubble generator (e.g. abubble dispersion device utilizing limewood splinters, or a ceramicsurface with pores).

In some embodiments, a surfactant may be used to stabilize the bubblesand prevent their coalescence. The surfactant may be an ionic or anon-ionic surfactant. The surfactant is used at a water solubleconcentration sufficient to prevent coalescence of the bubbles. Incertain embodiments the surfactant is a polyether. In particularembodiments, the surfactant may be PEG or PPG. The molecular weight ofthe PEG surfactant may be from about 100 to about 9000 daltons, forinstance about 100, 300, 400, 600, 900 1000, 1450, 3350, 4500, 8000 or9000 daltons. Alternatively, the surfactant may be PPG, and themolecular weight of the PPG may be about 340 to about 3500 daltons, forinstance about 340 daltons.

The bubbles may be produced by a means for creating and dispersingbubbles. For instance, a fine pore glass dispersion tube or sparger maybe connected to an aquarium pump and placed in a container holding amixture of embryos and debris in suspension. In certain embodiments, thebubble production and dispersion device comprises multiple point sourcesof bubbles, such as individual splinter of limewood. The splinters maybe inserted into a matrix, such as a length of silicone tubing, andoptionally coiled into a spiral and secured.

When embryos have been carried to a liquid interface by the bubbles,they may be removed from the surface froth by a suitable means, such asoverflow by gravity, or a skimmer or vacuum device. The device may beautomated.

Combination Device and Methods for Extracting and Separating Embryos

In further embodiments, the invention provides a combination device forextracting and separating embryos suitable for tissue culture. Theextractor may be in communication with the separator, or they may beoperated separately. The extractor comprises one or more fluid jets forextracting an embryo, such as a corn embryo, from a seed. The seed maybe a kernel on a corn cob. More than one fluid jet may be directed at asingle cob, and more than one cob may be simultaneously placed in theextractor.

After extraction of embryos from kernels, embryos and debris may fallinto or be conveyed to an embryo separator comprising, for instance, aflotation chamber. The means for conveying to and from the flotationchamber may be a screened conveyor belt or belts, or mesh that mayseparate debris from embryos via size-exclusion. The belt or mesh may bea woven or molded thermoplastic. Upon adding embryos to the flotationchamber, embryos are preferentially separated from other debris, forinstance via the described bubble method, and float to the surface wherethey may be removed via an opening or chute.

Thus, corn ears may be harvested, and embryos may be excised using afluid jet. The top of the kernel may be removed in order to facilitateembryo excision by the fluid jet, and the kernel contents aresubstantially removed from the ear by the jet. The fluid jet may be aliquid jet comprising the described excision media with an osmolality ofabout 0-600 mOsm/kg. In particular embodiments, the excision medium is asterile solution, for instance 0.1-0.2 M mannitol. Embryo as well asnon-embryo (e.g. endosperm) tissue may be extracted from the cob andkernel by the jet. Subsequently, embryos may be substantially separatedfrom non-embryo tissue, for instance by the disclosed separationmethods, and apparati, using shear forces including flotation ofembryos, by sieving, or the like. However, even embryos, or groups ofembryos, that are not separated from other tissue comprising a kernelmay be of use for subsequent tissue culture, including transformationand regeneration of transgenic plants.

If a flotation method is used for separation, a surfactant may be addedto the media in which the embryos are separated. The surfactant, such asPEG or PPG, reduces coalescence of bubbles, maintaining an effectiveaverage bubble size to promote efficient separation of embryos fromnon-embryo tissue, and the deposition of embryos at an air-liquidinterface where they may be harvested by fluid flow, by a skimmer, by avacuum device, or the like. By such methods, substantially excised andseparated plant embryos may be obtained, suitable for production oftransformed tissue and transformed plants.

Transformed Plants and Methods of their Production

The present invention also provides a transformed plant, such as a corn,cotton, or soybean plant, produced by the steps including (a) providingat least one transformable target tissue using one or all of the methodsor apparatuses described herein; (b) introducing a selected nucleic acidmolecule into the transformable target tissue to produce a transformedexplant; and (c) growing a transformed monocot plant from thetransformed explant. Preferred plants of the invention includetransformed members of the family Poaceae, including grain crops such ascorn (maize), millet, wheat, and rice; as well as transformed dicotplants such as cotton or soybean plants.

Particularly preferred monocot plants include transformed Zea species,such as Zea mays. Transformed corn preferably contains at least oneheterologous nucleic acid molecule capable of conferring a desired traitto the transformed corn, such as herbicide resistance, pest resistance,cold germination tolerance, water deficit tolerance, increasedproductivity, increased yield, and the like. Practical transformationmethods and materials for making transgenic monocot plants of thisinvention (for example, various media and recipient target cells,transformation of immature embryos, and subsequent regeneration offertile transgenic plants) are disclosed, for example, in U.S. Pat. No.6,194,636 to McElroy et al., U.S. Pat. No. 6,232,526 to McElroy et al.,United States Patent Application Publication Number 2004/0216189 toHoumard et al., United States Patent Application Publication Number2004/0244075 to Cai et al., which disclose methods useful with corn, andUnited States Patent Application Publication Number 2003/0024014 toCheng et al., which discloses methods useful with wheat, all of whichare incorporated by reference herein. Single or multiple heterologousnucleic acid molecules may be used for transforming the monocot plantsof the invention; for example, constructs for coordinated decrease andincrease of gene expression are disclosed in United States PatentApplication Publication Number 2004/0126845 to Van Eenennaam et al.,which is incorporated by reference herein. Numerous methods fortransforming other plants, including dicot plants, are well known in theart. For instance, methods for transforming soybean and cotton plantsare described in U.S. patent application Ser. No. 12/045,502 or U.S.Pat. No. 7,002,058, each of which are herein incorporated by reference.

In certain embodiments, for instance when an Agrobacterium- or otherbacterially-mediated genetic transformation method is utilized, theculture response or transformation frequency of embryogenic tissueisolated by the methods of the present invention may be enhanced by useof a co-culture medium comprising a bactericide such as carbenicillin,and/or 2,4-D at a concentration of about 0.5-1.5 mg/L. In a particularembodiment, the co-culture medium comprises the ingredients of Medium1898 of Table 9.

The seeds of resulting transgenic, fertile plants of the invention canbe harvested and used to grow progeny generations, including hybridgenerations, of transformed plants that include the heterologous nucleicacid molecule in their genome. Thus, the present invention includes bothprimary transformed plants (“R0” plants, produced by transformingembryos provided by a method of invention) and their progeny carryingthe heterologous nucleic acid molecule. Such progeny transgenic plantscan be prepared by crossing a transformed monocot plant of the inventionhaving the heterologous nucleic acid molecule with a second plantlacking the construct. Also, a transformed monocot plant of theinvention can be crossed with a plant line having other heterologousnucleic acid molecules that confers another trait to produce progenyplants having heterologous nucleic acid molecules that confer multipletraits.

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided.

“Embryo” is a part of a seed, consisting of precursor tissues(meristematic tissues) for the leaves, stem, and root, as well as one ormore cotyledons. Once the embryo begins to grow (germinate), it becomesa seedling plant.

“Meristem” or “meristematic tissue” consists of undifferentiated cells,the meristematic cells, which differentiate to produce multiple plantstructures including stem, roots, leaves, germline tissue and seeds. Themeristematic cells are the targets for transformation to obtaintransgenic plants.

“Explant” is a term used to refer to target material for transformation.Therefore, it is used interchangeably with “meristematic tissue” or“embryo” in the embodiments herein.

EXAMPLES

Those of skill in the art will appreciate the many advantages of themethods and compositions provided by the present invention. Thefollowing examples are included to demonstrate the preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. All references cited herein are incorporated herein byreference to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, or compositionsemployed herein.

Example 1: Method to Extrude Multiple Corn Embryos

This example describes a method using mechanical positive pressure froman extruder device to produce embryos suitable for tissue culture orgenetic transformation.

The tops of kernels were sterilely removed from an immature ear of corn(Zea mays) with a vegetable peeler. The peeler was pushed from the basalend of the corn ear to the apical end using a slight sawing motion toobtain a quick, sharp truncation of the kernels. While in this instancethe individual kernels are truncated to expose the interior tissues, inother embodiments, it may be necessary only to ensure that an opening(such as a puncture or incision or abrasion) is made in the pericarpwithout actual removal of pericarp material. Where intact embryos aredesired (for example, intact embryos for transformation), the size ofany opening is preferably sufficient to allow removal of the embryowithout damaging it. Opening of the pericarp can be accomplished byusing any suitable device, including, but not limited to, blades andabrasive materials. For example, a vegetable peeler is designed to berelatively safe and fast to use; it has a regulated cutting depth andalso requires less skill to use than a scalpel. Other tools with similarfunctions can be employed. The devices for opening the pericarp arepreferably sterilizable, for example, by autoclaving or heating or bychemical sterilization. These pericarp treatment processes can beautomated; for example, a blade or blades or abrader can be motorized.

A sterile extruder (in this case, a 4 millimeter diameter rod) waspushed against the base of the truncated kernels. Other suitableextruding devices may be employed. Preferably, such devices should havea size and shape capable of applying a relatively localized force to thebase of the truncated kernels to eject the embryos and endosperms.Preferably, the force applied is of sufficient magnitude and is appliedin a suitable direction such that the advancing extruder does not “rideup” over the forward kernels. The trailing edge of the extruderpreferably also provides a surface on which the ejected embryos andendosperms accumulate; for example, a flat piece of stainless steel witha rounded front edge could be used. In this example, the embryos weregently squeezed out from the pericarp, followed by the endosperms. Theextruded embryos and endosperms came to rest on the top of the advancingextruder rod, and were not crushed during the process.

The mixture of embryos and endosperms was washed with an aqueous fluidmedium (e.g. water, liquid medium, or saline) onto a sterile mesh havingdiamond-shaped openings (about 2×3 millimeters). The endosperms wereobserved to be largely retained, and the smaller embryos and somesmaller endosperm debris were washed through the screen into acollecting receptacle. The collected embryos were washed twice to removesmall debris.

The washed embryos were further purified by a flotation process. In thefirst step of the floatation process, the aqueous fluid medium wasthoroughly withdrawn from the collecting receptacle, which was allowedto dry briefly (for example, about a minute), such that remainingaqueous medium withdrew from the waxy surface of the embryos, exposingthem directly to the air. New aqueous medium was added, and the majorityof the embryos floated because their waxy surface was not rewetted bythe fluid. Non-embryo tissues such as endosperm debris remainedsubmerged in the medium, and a clear separation of embryos andnon-embryo tissues was obtained. The floatation of the extruded embryoscould be improved by more rapid, complete, or reproducible withdrawal ofthe aqueous medium, such as through the use of aspiration, or bycapillary action (e. g., use of a sterile absorbent placed in thecollecting receptacle to absorb the fluid away from the extrudedembryos).

A yield of approximately 100 embryos was isolated in this preliminaryexperiment, wherein only a portion of the embryo-endosperm material fromthe entire ear was processed. These results demonstrate that methods ofthe present invention are practical and convenient for harvesting largenumbers of immature embryos from corn cobs.

The embryos isolated by a method of the invention may then be used intissue culture procedures, for example, regeneration methods to generatetransgenic corn plants. Transfer of the isolated embryos to culturemedium was easily done by placing forceps, with the tips closedtogether, underneath the floating embryos, lifting them free of theliquid with the forceps and placing them on culture medium. Anothertechnique could be to pick the isolated embryos up with an instrumentthat has a hydrophobic surface. An additional technique would be totransfer embryos by hydrophobicity, for example, transferring them tothe medium surface by a small puff of air or sudden mechanical movement,such that their kinetic energy exceeds the hydrophobic force that holdsthem to the instrument.

Example 2: Visual Confirmation of Embryo Size

This example describes an improvement to one embodiment of the method ofthe present invention, as described in Example 1. Using the approachdescribed in Example 1, immature corn embryos need to be as close to thetruncated part of the kernel in order to be ejected in the greatestnumbers. Variation in immature corn embryo size is an importantconsideration in gauging the amount of kernel top to remove. Embryostend to be largest in the mid-section of the ear, with somewhat smallerembryos towards the ends. Smaller embryos, e. g., smaller than about 1.5millimeters in length are more difficult to remove unless they are closeto the truncation.

One way to ensure that enough of the kernel has been decapitated aboveembryos of varying sizes is to observe the cob during the decapitationprocess under low magnification. For example, low magnification goggles(e.g. Donegan Opti-VISOR headband binocular magnifier equipped with aNo. 7 lens, which provides a 2.75× magnification) were used to aidvisual confirmation of embryo size and suitable truncation of thepericarp. If the first cut did not remove enough of the kernel apex, asecond cut could be made. Other low magnification devices, using thesame or similar magnifications could be used. For example, availablelenses for the Opti-VISOR provide magnification ranging from 1.5 to3.5×.

Example 3: Extrusion of Embryos and Endosperms

This example describes an improvement to one embodiment of the method ofthe present invention, as described in Example 1. Powered devices may beused to assist in the extrusion of embryos and endosperm. For example, apower chisel such as a WeCheer 320 power chisel (WeCheer Industrial Co.,Taichung Hsien, Taiwan, R.O.C), fitted with a rounded extruder device,can be used to reduce the force a person needs to exert to eject theembryos and endosperms. Other powered devices are available and can besimilarly used. Preferably, the “chisel” portion of such a tool (or anypart of the tool that might come into contact with the embryos) can beconveniently sterilized, for example, by insertion into a beadsterilizer.

In one experiment, the blade of a stainless steel weighing spatula wasbent back on itself to provide an extruder device having a roundedleading edge. After insertion into a WeCheer 320 power chisel, a portionabout 10 centimeters long extended out from the power chisel's chuck.This assembly was used to eject the embryos and endosperms fromindividual rows of decapitated kernels. As the extruder device (modifiedspatula) moved down a row of kernels, a slight tendency for the spatulato slide off center to the left or right was observed; however, thistendency could be corrected by including a small keel-like extension ofthe spatula on each outer edge.

Example 4: Mechanized Embryo Extrusion

This example describes an improvement to one embodiment of the method ofthe present invention, as described in Example 1. Mechanization of theembryo extrusion process can be achieved by use of a suitable device,such as, but not limited to, the device described herein andschematically diagrammed in FIG. 1 . This device includes two motors.The first motor (D) is a stepper motor that can rotate the corn ear (A)so that new rows of kernels are exposed to the two extrusion rods (G),which apply force to squeeze the embryos and endosperms out of theirpericarps.

Rods (G) are conveniently located on opposite sides of the ear in orderto balance the pressure applied to the ear relative to the ear'slongitudinal axis. However a single rod can be used, or more than tworods; where multiple rods are used, it is preferable that they arepositioned so as to evenly distribute the resulting mechanical pressurearound the ear. The rod need not be a straight rod; in one embodiment ofthe device, a flexible “collar” encircling the circumference of the earis used instead of a rigid rod. In another embodiment, multiple shortrods or rollers are arranged in a flexible, circular configuration thatcan be slid along the ear's longitudinal axis, applying mechanicalpressure to many or all rows of kernels simultaneously.

The second motor is connected to the pinion gear (E) connecting to arack (F) so that up and down linear motion of the ear occurs. The baseof the ear is held firmly to a handle (B) by means of a screw extendingfrom the handle down into the base of the ear.

The narrowed middle portion of the handle is square so that it will notrotate unless the holder (C) to which it is attached is rotated by thestepper motor (D).

Before insertion into the machine, the tops of the kernels aredecapitated as in Example 1 so that the embryos and endosperms can besqueezed out. To start the process, the ear is lowered until the tworods (G) are near the base of the ear just below the handle (B). Thenthe rods are pressed against both sides of the ear and the rack andpinion assembly draws the ear upward. As this happens, the embryos andendosperms are removed from a couple of rows, fall downward into thecollection dish (H) resting on the base (I), and collect in a pile (J).When the rods approach the apical end of the cob, the cob is withdrawnupward to its original starting position and rotated slightly by thestepper motor until new rows of kernels come into position.

Various degrees of automation of this machine are possible, includingsensors to automatically adjust the vertical starting and finishingpositions as well as the rotary start and finish positions. A rack andpinion is not the only method by which linear motion can be obtained.Pneumatics or hydraulics may be preferred for some applications. Rods(G) can be automatically opened by a suitable mechanism. When a new earis loaded, it may be preferable to raise the ear to a position highenough to clear the rods.

Example 5: Hydrophobic Separation of Embryos

This example describes a modification to one embodiment of the method ofthe present invention, as described in Example 1. In separationapplications the material of interest frequently appeared at theinterface of dissimilar phases (for example, between aqueous andlipophilic solvents). Removing the material of interest from such aninterface can pose problems, and has in the past been a manual processinvolving close contact with the extractant and the material to beextracted. Often the only way to successfully separate out a componentis to use a material of the same polarity orhydrophobicity/hydrophilicity. In the case of immature corn embryosextruded by a method of the invention, the embryos are found at theaqueous/air interface. The corn embryos' surface is waxy, i.e.,lipophilic or hydrophobic, and when an embryo cuticle is contacted witha substance of similar hydrophobicity, the embryo will tend to stick tothe hydrophobic surface. The embryo's hydrophobicity reduces the surfacetension of the water around it, which helps the embryo to “float” at thesurface of the aqueous/air interface.

One approach that takes advantage of these physical characteristicswould be to touch the floating embryos with a hydrophobic material suchas hydrophobic filter paper, e. g., Whatman No. 1 PS paper, which is awater-repellant phase separating paper impregnated with silicone(Whatman plc, Brentford, Middlesex, U.K.). In one example, a piece ofsterile hydrophobic filter paper can be lowered onto an entire containerof floating embryos and pick them all up at once. In another example, asmall piece of the hydrophobic paper can be used to successively pick upa number of embryos and transfer them to the next container. In a thirdexample, either a small piece of the hydrophobic paper or a hydrophobicpipette tip would be used to contact and pick up individual embryos andthen dispense them with a puff of air from the pipettor. Ordinarypipette tips could also be modified for such use by inserting a pipettetip into a short length of hydrophobic tubing (for example, siliconetubing); the embryo could then be picked up by hydrophobic attraction tothe distal end of the hydrophobic tubing, and then released bydispensing a puff of air from the pipette. Reduced surface tensionaround the hydrophobic embryos helps them float on an aqueous surface,and the floating embryos could also be transported by moving them on theaqueous surface (for example, by an air jet directed at the embryos).Picking up and dispensing of embryos can be automated usingmodifications of existing devices, such as machines designed for colonypicking or for retrieving protein spots on stained 2-dimensional proteingels.

Example 6: Further Methods of Ejecting or Extruding Embryos

The method of the present invention encompasses the use of various typesof force, or combination of forces, for separating the embryo from itsseed. This example describes further embodiments. In one basic method asdescribed in Example 1, mechanical positive pressure is applied to thebase of a truncated seed (such as a corn kernel) to eject the embryo outthrough the truncated top of the seed.

In another embodiment, centrifugal force can be used to eject theembryo. For example, a corn ear (the kernels of which have previouslybeen truncated) could be spun about its longitudinal axis at a speedsufficient to eject the embryos and/or endosperms in a radialtrajectory. Spinning could be achieved by any suitable technique, suchas, but not limited to, contacting the apical end of a corn ear with afreely rotating cone, wherein the rotation of the ear is kept within alimited longitudinal range, for example, by attaching the basal end ofthe ear to a handle which is then inserted in a holder within which itcan rotate. In one exemplary embodiment using centrifugal force, about athird of the top of each kernel on a corn ear was removed with ascalpel, and the ear rolled on a surface to loosen the embryo andendosperm within the kernels. The ear was snapped into two pieces, eachabout 750 millimeters in length. Each piece was placed in a250-milliliter centrifuge bottle with about 100 milliliters of water.These were centrifuged 15 minutes at 5000 rpm to eject the embryos.Examination of the ears after centrifugation showed that, in someportions of the ear, all the embryos had been removed by thecentrifugation, whereas in other areas, few or no embryos were removed.The ejected material was centrifuged and the supernatant removed toleave a slurry, which contained intact embryos (estimated to includeabout 20 percent of the total number of embryos). In another example, animmature ear of corn is harvested (typically between about 10 to about14 days post-pollination). The ear is disinfested, and under sterileconditions the top of each kernel is cut off. The ear is mounted on adrill bit on an electric drill (or a similar device) and the ear issurrounded by a large sterile collection vessel (e. g., a large glassbeaker). The ear is spun at a rotation sufficient to eject the immatureembryos, and the ejected tissues are collected from the sterilecontainer. Immature embryos are collected, for example, by manualcollection, or by rinsing the container with sterile tissue culturemedium and recovering an enriched fraction containing the embryos (e.g., by sieving, by the use of a liquid density gradient, or by othermethods to separate embryos from non-embryo tissues as describedelsewhere in this disclosure). The immature embryos (or callus derivedfrom the immature embryos) can be used subsequently for transformation.Improved results using these and other centrifugation methods can beobtained by determining preferred centrifugation times and speeds byroutine testing.

Another embodiment employs bulk maceration of kernels. An immature earof corn is harvested (typically between about 10 to about 14 dayspost-pollination). The ear is disinfected. The pericarp can be openedunder sterile conditions or the kernels can be left intact. The kernelsare removed from the cob by any suitable procedure, including, but notlimited to, using a scalpel or other bladed tool. The kernels, onceseparated from the cob, are placed in tissue culture medium. Thekernel-medium mixture can be subjected to further tissue disruptionusing a suitable cutting device, such as, but not limited to, a blender.Immature embryos are collected, for example, by manual collection, or byrinsing the container with sterile tissue culture medium and recoveringan enriched fraction containing the embryos (e. g., by sieving, by theuse of a liquid density gradient, or by other methods to separateembryos from non-embryo tissues as described elsewhere in thisdisclosure). Immature embryos (or callus derived from the immatureembryos) can be used subsequently for transformation.

In a further embodiment, fluid jets (of gases or liquids or combinationsthereof) could be used to dislodge embryos. One example of this approachis to automatically rotate a corn ear in a stepwise or continuous(helical) manner past a stationary jet, collecting the ejected materialcontaining the embryos and further isolating the embryos if necessary,for example, by size separation on a mesh or screen or the like. Wherethe corn ear is vertically orientated (with respect to its longitudinalaxis), it may be preferred to rotate the ear in an upward helicaldirection, or otherwise move the ear relative to the jet so thatextracted embryos tend to wash downward.

In yet another embodiment, linear deceleration or linear accelerationcould be employed to dislodge or eject the embryos. For example, a cornear could be administered a shock parallel to the ear's longitudinalaxis and of sufficient force to eject the embryos and endosperms. A cornear could be enclosed in a suitable sterile, high impact-resistantholder, which could be subjected to sudden acceleration or deceleration,for example, by a sharp impact (e. g., as from a mallet).

Another improvement to the method would be to facilitate ejection orextrusion of the embryo from the truncated seed. For example, embryoscould be loosened or dislodged within their native position within theseed by applying a force to the tops of intact seeds (e. g., by applyinga roller or other means of applying pressure to the tops of rows of cornkernels in an intact ear or rolling or pressing the ears themselves on asurface prior to decapitating the tops of the kernels). Embryos may alsobe loosened within the seed by application of vibration, for example, byultrasound. Another approach would be to remove additional non-embryotissue, such as additional lateral wall (pericarp) material, beforeembryo ejection or extrusion. For example, a V-shaped knife or otherinstrument could be used to remove some of the lateral walls of cornkernels in rows in the ear.

Example 7: Automated Embryo Isolation Using Fluid Jet Positive Pressure

This example describes a further embodiment of the present invention. Inthis example, an automated device uses fluid jet positive pressure todislodge embryos from seeds. With reference to FIG. 2 , a roboticgrasper C (preferably capable of motion in three dimensions by means ofrobot A and motor B, or by an equivalent means) picks up a corn ear I(by a handle D having a baffle E) in a defined position for a rack onthe robot deck. The robot inserts the corn ear into tube H (optionallymade of transparent material for ease of visual observation) at astarting position below flange F. Fluid jet positive pressure isintroduced through aperture G and the ear is simultaneously raised (inthe Y dimension) and rotated by robot A and motor B respectively,preferably resulting in each kernel being struck by the fluid jet,causing the embryo and endosperm to be dislodged. The fluid passingthrough aperture G can be at least one gas, at least one liquid or anycombination thereof. The fluid jet can exert force continuously ornon-continuously, for example, as in pulses. As the embryos andendosperms are dislodged by fluid jet positive pressure from aperture G,they fall down to the shaking screen J, which retains the endospermswhile permitting the embryos to fall through to the collecting surface K(for example, sterile cheesecloth) below. Excess fluid can be optionallycollected in waste or recycling receptacle L. After completion of theembryo removal process for each corn ear, the interior of the tube canbe briefly washed down manually or by automated jet above or below theflange F.

Example 8: Methods of Processing Crude Embryo Preparations

The embryo preparations obtained by methods such as those described inExamples 1 through 7 may include both intact embryos and partialembryos, which may be accompanied by non-embryo tissues, such asendosperm and glumes. Some applications may not require furthertreatment or separation steps, for example, in a mass transformation ofsuch a “crude” embryo preparation where embryos (intact or partial) neednot be separated from non-embryo tissue. For example, callus derivedfrom either intact or partial immature corn embryos can be used fortransformation, regeneration, and production of fertile, transgenicplants. Thus, both intact and partial embryos may serve as transformableexplants and need not be separated from each other. However, in othercases it may be desirable to further purify embryos from a crude embryopreparation.

Procedures wherein some difficulties may be encountered in processingcrude embryo preparations include: (1) rinsing away of non-embryo tissue(e. g., cell debris, starch grains, undesirable proteins), (2)efficiently removing excess liquid from embryos after extrusion orrinsing using liquid, and (3) adding liquid with minimal turbulence sothat the embryos float and do not become submerged.

A porous material is useful for separating non-embryo tissue fromembryos. Any suitable porous material can be employed, preferably havinga mesh or hole size small enough to retain embryos but let smaller,non-embryo tissues or debris pass through, and capable of beingsterilized (e. g., by autoclaving, heat, irradiation, or chemicalsterilization). Suitability of materials is easily judged or tested bysimple experimentation by one skilled in the art. Examples of suitablematerials include cheesecloth or other woven material, and other meshesor screens. In some embodiments, perforated solid materials can be used,including perforated ceramics, polymers, metals, or glasses (forexample, in the form of a Büchner or similar separatory funnel).Cheesecloth of appropriate gauge, for example, has a mesh size smallenough to retain embryos but allows smaller debris to pass through, andis autoclavable. Cheesecloth can be attached to a frame or collar (forexample, the frame holding embryo collecting surface K in FIG. 2 anddescribed in Example 7) to allow the cheesecloth and all the retainedembryos to be simultaneously submerged for easy rinsing. For example,cheesecloth can easily be attached to the frame by means of an elasticband or the like (e. g., silicone tubing); such frames are easilymanufactured, for example, from a beaker or graduated cylinder made ofautoclavable material (e. g., polypropylene, polymethylpentene,polycarbonate, or autoclavable glass) cut into sections. Cheesecloth hasstrong capillarity, allowing liquid to be efficiently pulled away fromthe embryos, thus exposing their waxy epidermis to air prior toflotation. In the flotation step, the cheesecloth is simply submerged inaqueous liquid, allowing the embryos to float off.

Example 9: Substantial Isolation of Embryos Using a Fluid Jet

This example describes a further embodiment of the present invention. Inthis example, multiple embryos were dislodged from seeds by fluid jetpositive pressure. In the simplest example, a 200-microliter pipette tipwas attached to a vertical sink nozzle with Parafilm®. When the tapwater was turned on a jet emerges from the pipette tip with considerableforce. The tap water pressure was estimated to be about 60 pounds persquare inch. This fluid (liquid) jet was trained on an immature corn ear(contained in a beaker) wherein the kernels had been decapitated asdescribed in Example 1. As the jet stuck each kernel, the endosperm andembryo were ejected, and collected in the beaker. Since the endosperm atthis stage is a relatively soft tissue it was fragmented into manysmaller pieces by the jet, whereas the embryos appeared to remainintact.

The endosperm and embryo tissue dislodged by the jet was poured directlyonto a No. 60 cheesecloth (other suitable porous material, such ashydrophilic mesh of the appropriate mesh size, could be substituted).Different “grades” of cheesecloth are available (for example, grades 10,20, 30, 40, 50, 60, 70, 80, and 90, where the mesh openings decreasewith higher grades), and the grade or mesh size appropriate to theaverage size and shape of a given type of embryo is easily selected bysimple experimentation. The embryos and larger fragments of theendosperm were retained on the upper surface of the cheesecloth. Priorto the next step, the cheesecloth was allowed to partially dry bywicking away excess liquid. This pulled liquid away from the tissues andexposed the surfaces of the embryos to air. When the cheesecloth waslowered into aqueous liquid, the embryos floated because their waxyepidermis did not rewet.

In a simple set up, the cheesecloth (or other suitable porous material)can be manually stretched or held over a receptacle or waste containeras the liquid holding the crude embryo preparations is poured throughthe cheesecloth. For sterile work, the cheesecloth can be attached torigid frames, which can be autoclaved before use. Snap-together sieveswith handles, such as those available in kitchen supply stores, couldalso be used in the method.

Example 10: Devices for Embryo Extraction Using a Fluid Jet

This example describes various embodiments of an apparatus formechanically preparing multiple corn embryos suitable for tissueculture.

One embodiment includes an apparatus for preparing multiple corn embryosusing a fluid jet, generally similar to the device depicted in FIG. 2 .A transparent, open-ended cylinder was made by cutting the ends off a1-liter autoclavable polymethylpentene (PMP) graduated cylinder. Apipette tip (e.g. 1250-microliter Gilson DistriTip®, tapered to avoidbackpressure build-up) was secured to the side of the cylinder andserved as an aperture for guiding a fluid stream as a jet through a holemade in the cylinder's wall. Fluid (in this case, water) was fed throughthe pipette tip from PharMed® high pressure autoclavable peristalticpump tubing. The water was delivered from a laboratory sink tap, butcould be an aqueous fluid delivered from a pump or other source. Using apump capable of delivering a sterile fluid is preferable when, forexample, sterile culture medium or a sterile salt solution is found tobe superior to water as a liquid for substantial isolation of embryos.An example of a suitable pump is a Masterflex® pump with the highpressure L/S pump head (Cole-Parmer Instrument Co., Vernon Hills, Ill.),which can deliver sterile liquid at up to 100 psi when used with highpressure tubing.

A corn ear with previously decapitated kernels was manually positionedwithin the cylinder. Once the ear was positioned appropriately withinthe cylinder, each kernel was subjected to positive pressure from thewater jet. This resulted in the embryos and non-embryo tissues beingextruded from the kernels. Examination of the ear after this treatmentindicated efficient removal of the embryos from the kernels. Theextruded material was washed down the cylinder's interior walls to anembryo collector positioned beneath the cylinder. The embryo collectorincluded: (1) a coarse plastic screen (onto which larger debris wastrapped), heat-fused to the cut-off top of a Tri-Pour™ plastic beakerand stacked above (2) a finer screen (Grade 60 cheesecloth, onto whichthe extruded embryos were trapped), secured with an elastic band to thecut-off top of a second Tri-Pour™ plastic beaker and stacked above (3) awaste collection beaker or other container (in which the fine debris,non-embryo tissues, and waste liquid was collected).

Modifications to these and similar embodiments are easily made by oneversed in the art. For example, with regard to positioning the corn earor seed for application of the fluid jet, the ear could be held manuallyin place, or preferably, mounted securely within the cylinder by amovable support capable of moving the ear in three dimensions. Forexample, the ear could be mounted to a threaded metal or polymer rod,such as a polypropylene rod, which could be used to move the ear alongits longitudinal axis as well as to rotate the ear). Another example ofa mounting mechanism is depicted in FIG. 3 , which illustrates amagnetic “handle” by which an ear can be secured to a robot arm.

In other embodiments, however, the corn ear or ears need not beindividually secured to a holder but can be freely movable so as toallow multiple kernels to be contacted by the force used to remove theembryos from the kernels. For example, at least one ear, or multipleears, can be borne on or held between at least one support, such as, butnot limited to, at least one plane, frame, grid, screen, mesh, platform,roller, guide wire or rod, and belt, wheel, or roller conveyor. Such asupport could be movable or could cause the ear or ears to move, forexample, by vibration, rolling motion, gravity, or other mechanisms.Substantially isolated embryos could pass through the platform itself ifthe platform was porous (e. g., made of mesh). The ear or ear can alsobe floated on a fluid in a manner allowing each ear to rotate orotherwise move freely while afloat. The fluid, such as a liquidcontaining the substantially isolated embryos, could be continuallydrained off, optionally through a filtering or sedimenting device, orcollected for centrifugation.

Devices for obtaining motion along the longitudinal axis of a corn earinclude, but are not limited to, ball screw-driven slides or belt-drivenslides, such as those commercially available from various manufacturerssuch as Techno, Inc. (New Hyde Park, N.Y.; techno-isel.com). To obtainrotary motion for rotating a corn ear, a stepper motor can be used, forexample, a stepper motor attached to a slide plate. Rotary motion canalso be provided by rolling devices, for example, by parallel round ortubular rollers between which the corn ear is held and rotated.

The shape of the fluid jet can be advantageously modified according tothe desired application. For example, a narrow column-shaped jet ofuniform diameter is useful for removal of embryos from one seed at atime. Where it is desirable to increase the rate at which embryos aresubstantially isolated, multiple embryos can be simultaneously removedfrom their seed by a fluid jet; this can be achieved, for example, byusing at least one single fluid jet that covers a larger area, or byusing multiple jets simultaneously. In one embodiment, multiple jets,such as multiple parallel, narrow, column-shaped jets (for example,produced by multiple nozzles similar to that used in Example 9 andoptionally connected to each other by a manifold) are used to directfluid jet positive pressure on multiple seeds to substantially isolatetheir embryos substantially simultaneously. Automation of these andother devices can further include optical or mass sensors to aid inpositioning the ear and fluid jet relative to each other.

In another embodiment, at least one fluid jet that covers a larger area(for example, wherein the fluid jet simultaneously impacts multiplekernels, or multiple rows of kernels on a corn ear) can be used. Thedimensions of such a jet preferably allow the jet to enter the kernelsand wash out the embryo. Typically, corn embryos used in geneticexperiments are immature and generally in the size range of about 1.8 toabout 2.2 millimeters in length; the kernels holding these immatureembryos are generally in the size range of between about 4 and about 5millimeters in width. For embryos of this size, an appropriate fluid jetcan be, for example, between about 0.5 to about 1 millimeter in width.

Any suitable means for producing such a larger fluid jet may be used,such as, but not limited to nozzles that generate non-columnar fluidjets. Examples of suitable nozzles include, but are not limited to,nozzles that generate a flat spray pattern and nozzles that generate afan- or a cone-shaped spray pattern. In one example, a commerciallyavailable flat spray nozzle (number 23990-1/4-04, Spraying Systems Co.,Dillburg, Pa.) was used with a Masterflex® L/S pump (model 77250-62) topump liquid at 1 liter per minute and 30 psi; embryos were excised froma corn ear under these conditions. Another example of a preferred nozzleis a nozzle that generates a fluid jet in the form of a flat “sheet” offluid, such as is depicted in FIG. 4 . Such a nozzle preferably iscapable of generating a uniform, flat fluid jet that maintains acoherent, uniform sheet-like flow for at least a distance sufficient toallow the flow to contact more than one seed (and preferably severalseeds) at the same time. The novel nozzle depicted in FIG. 4 is designedto generate a uniform, flat sheet-like jet that is about 0.5 to about 1millimeter in thickness, greater than about 20 millimeters in width, andmaintains the sheet-like flow over a distance of about 15 to about 30millimeters from the nozzle's aperture. This latter distance permits thejet to be moved along the rows of kernels with minimal adjustment neededfor differences in distance between the surface of the kernels and thenozzle's aperture.

Regardless of the area or shape of the jet or spray pattern generated bythe nozzle or aperture through which the liquid flows, nozzles orapertures are preferably used with flow rates and pressures sufficientto generate enough fluid force to dislodge the embryo from its seed,without damage to the embryo. In some embodiments, it is preferable touse a lower flow rate and possibly a higher pressure, to minimizeconsumption of fluid (such as medium) as well as to minimize the wastegenerated.

Example 11: Using a Gas Jet to Substantially Isolate Embryos

This example describes further embodiments of methods and devices formechanically preparing multiple corn embryos suitable for genetictransformation or tissue culture. As described in Example 6, gas jetscan also be used for the substantial isolation of multiple embryos. Anapparatus similar to that described in Example 10 was modified for usewith gas. A 1-milliliter pipette tip (Marsh Bio Products-ABGene,Rochester, N.Y.; catalogue number TN1300RS,) was secured to the side ofthe one liter polymethylpentene graduated cylinder and served as anaperture for guiding a stream of air as a jet through a hole made in thecylinder's wall. Air was supplied from a compressor pressurized tobetween about 60 to about 100 psi. An air valve for convenience waspositioned in line between the compressor and the pipette tip. The airjet emerging from this pipette tip was used to dislodge the embryos froma prepared corn ear. Examination of the kernels after they had beensubjected to the air jet showed that the thick pericarp remained inplace and surrounded by papery glumes, and the pericarp contents (embryoand endosperm) had been removed. Examination of the tissue retained bythe grade 60 cheesecloth showed that this included dislodged embryos aswell as some glumes dislodged by the high-pressure air jet. The glumesof corn have a waxy surface like the embryos and also float followingthe flotation procedure. Using lower air pressures can reduce glumecontamination.

Example 12: Substantial Isolation of Embryos Using other Fluid Forces

This example describes further embodiments of methods and devices formechanically preparing multiple corn embryos suitable for genetictransformation or tissue culture. Forces exerted by fluids, other thanpositive fluid pressure from a fluid jet, can be used to substantiallyisolate embryos. In one experiment, the tops of kernels were removedfrom a corn ear, which was placed inside a bottle containing steriledistilled water and shaken vigorously by hand. This resulted in thesubstantial isolation of 90 out of the ear's 200 embryos. Anotherexperiment repeated the preceding procedure except that the shaking wascarried out in a mechanical paint shaker. In this experiment, 56 embryoswere substantially isolated out of the ear's 190 embryos. In a thirdexperiment, a similar procedure was carried out, except that the cornear was pre-soaked in 211 medium (1 liter: N6 Basal Salt Mixture fromDuchefa: 1 pkg (i.e. 3.952 g) (Gold Biotechnology Inc, St. Louis, Mo.,U.S.A.); 2,4-D (1 mg/ml): 1 ml; Thiamine (0.5 mg/ml): 2 ml; NicotinicAcid (0.5 mg/ml): 1 ml; L-Asparagine monohydrate: 0.91 g; Myo-inositol:100 mg; MES: 0.5 g; MgCl₂6H₂O: 1.6 g; Casein Hydrolysate: 100 mg;Proline: 0.69 g; Sucrose: 20 g; Agar: 2 g, pH 5.8), and the shaking wascarried out in a paint shaker. In this experiment, 109 embryos weresubstantially isolated out of the ear's 210 embryos. In these cases,non-jet fluid force from movement of the liquid around the corn earresulted in the substantial isolation of the embryos; the fluid forcecould include fluid turbulent flow, fluid laminar flow, shear from fluidflow, negative fluid pressure (for example, resulting in cavitation), orcombinations thereof. Forces can also include forces generated byacoustic techniques, such as by an acoustic wave or waves (pulsed orcontinuous) in either gas or fluid phase.

The preceding examples (including Examples 9-11) described use of afluid jet to remove embryos from an immature ear. During theseprocedures, it was observed that the fluid jet generally also caused atleast part of the endosperm to be released from the kernel. Theendosperm tissue was observed to be softer and more friable than theembryos, and tended to disintegrate to varying degrees (in contrast tothe embryos, which tended to remain intact). It is possible that theendosperms disintegrate upon exposure to shear caused by the fluid jet.This shear is believed to be non-uniform, resulting in the variabilityin disintegration observed; nonetheless, a large proportion of theendosperm material that was sufficiently disintegrated to pass throughthe cheesecloth, leaving a retentate made up of a semi-pure preparationof embryos.

When a low-pressure jet from an ordinary laboratory squirt bottle wasdirected at the cheesecloth retentate, more of the remaining endospermtissue was disintegrated further and washed through the cheesecloth,leaving behind a relatively more pure preparation of embryos. Thus it isreasonable to predict that if the retentate is uniformly exposed to ashear force of the correct intensity, all or substantially all of theremaining endosperm should disintegrate and pass through thecheesecloth. Such a shear force could be generated by any suitablemeans, such as, but not limited to, a single jet, multiple jets, asheet-like or curtain-like jet, rapidly moving jets, and acceleration ordeceleration of the endosperms. Additionally, if the jet used toinitially release the kernel contents is designed to expose a higherproportion of the endosperms to shear during ejection, an initial higherpurity embryo preparation could be obtained.

A non-limiting embodiment for applying shear to further purify embryosfollows. Once the embryos and partially disintegrated endosperms arereleased from a cob, the remainder of the endosperm can be rapidlyfragmented by fluid flow, for example, from a spray nozzle, that strikesthe endosperm uniformly and simultaneously. One suitable type of nozzleis a full cone nozzle. Full cone nozzles generate a spray patterncompletely filled with drops. An internal vane within the nozzle impartscontrolled turbulence to the liquid prior exiting to the orifice,allowing formation of the spray pattern. Commercially available nozzleshave spray patterns that are round, square, or oval. An example of asuitable full cone nozzle is known as “UniJet® Spray Nozzle, StandardSpray, Small Capacity” (Spraying Systems Co., Wheaton, Ill.; part numberTG-SS0.3).

Example 13: Devices Using a Combination of Forces

This example describes several additional embodiments of the method ofthe invention, which use a combination of forces to substantiallyisolate multiple embryos from seeds.

FIG. 5 illustrates a device using a larger fluid jet (as described inExample 10). This device includes a nozzle for generating a fluid flowsuch as a larger fluid jet (for example a flat fluid jet), and,optionally, a suction head, or component for applying negative fluidpressure (e. g., by vacuum or suction), for dislodging embryos and/orfor collecting the dislodged embryos. FIG. 5A (top) depicts across-sectional view of an example of such a device, showing how thenozzle, optional suction head, and corn ear can be positioned relativeto each other. The corn ear, nozzle, and optional suction head can bemoved relative to each other; for example, the corn ear may bestationary while the nozzle and optional suction head are moved, or thenozzle and suction head may be stationary while the corn ear is moved.FIG. 5B (bottom) schematically depicts a corn ear positioned in thedevice, and shows the nozzle positioned to generate a flat fluid jetwherein the jet impacts multiple kernels in a row.

FIG. 6 depicts an embodiment of a suitable suction head or component forapplying negative fluid pressure (e. g., by vacuum or suction), such asis optionally used in the device of FIG. 5 , and which can also be usedon its own to substantially isolate embryos. The suction head caninclude one or more apertures through which negative fluid pressure canbe applied. The suction head can also include a means for dispensingfluid (such as gas or liquid, e. g., water or medium), for example,multiple apertures in the suction head. For use with corn, the suctionhead is preferably shaped to follow the contours of a typical corn ear,and is preferably capable of entrapping embryos from multiple kernels orfrom multiple rows of kernels. It is envisioned that the suction headcan be manufactured of a rigid material (such as stainless steel orother metals), or of a flexible material to allow easier conformation ofthe suction head to the contours of a corn ear, or of combinationsthereof. Embryos can be substantially isolated by any combination ofmechanical positive pressure (exerted, for example, by a leading edge ofthe suction head), negative fluid pressure (e. g., suction or vacuum),and fluid force (such as, but not limited to, positive pressure from afluid jet, fluid turbulent flow, and fluid laminar flow entrappingmaterial from the interior of the kernel)

Devices for applying force for substantially isolating embryos, such asare described in Examples 1, 3, 4, 6, 7, 9, 10, and the present example(including, but not limited to the devices illustrated in FIGS. 5 and 6) can be moved relative to the corn ear. The ear may be stationary, orthe device may be stationary, or both can be moved. Because corn seedtypically occurs in relatively uniform rows arranged parallel to thelongitudinal axis of the corn ear, the device is typically moved(relative to the ear) so that the device passes parallel to thelongitudinal axis of the corn ear and following a row or multiple rowsof kernels. However the motion of such devices relative to the ear canfollow the circumference of the ear, or can be random, or can be anycombination of suitable motions.

FIG. 7A through 7C depict different views of an embodiment of a devicethat uses a combination of forces to substantially isolate multipleembryos from seed (in this example, corn). This device includes a headwith a leading edge capable of applying a predefined amount ofmechanical pressure to the base of kernels that previously have had thepericarp opened or truncated, so that the embryos are extruded from thekernels in a manner similar to those described in Examples 1, 3, and 4.The device further includes a component for applying negative fluidpressure (e. g., by vacuum or suction) for dislodging embryos and/or forcollecting the dislodged embryos. The extruded embryos (and accompanyingnon-embryo tissues) are thus separated from the corn ear and can becollected by application of negative fluid pressure. The collectedembryos and non-embryo tissues can be further separated, if desired, bysuitable means, such as by size-separation, hydrophobic separation, ordifferential centrifugation. A variation of this device could include ameans for dispensing fluid (such as liquid, e. g., water or medium), forexample, multiple apertures in the suction head.

The embryo extraction devices depicted in FIGS. 5, 6, and 7 aredescribed as illustrative examples that are not intended to be limiting.These and other such devices can include additional components, forexample, means for separating the embryos from non-embryo tissues orfrom fluids used in the substantially isolation process.

Example 14: Viability Data

The multiple monocot embryos provided by use of the methods and devicesof the present invention are most preferably embryos suitable forgenetic transformation or tissue culture application such astransformation and regeneration of plants. This example furtherillustrates the utility of methods of the invention in providingmultiple monocot embryos that are viable and suitable for genetictransformation or tissue culture. In this example, the quality ofimmature corn embryos obtained by different excision methods wascompared in their response to transformation by Agrobacteriumtumefaciens.

Corn embryos excised using this apparatus and method were transformedusing standard methods known in the art of corn transformation. (e.g.Cai et al; U.S. Patent Application Publication 2004/0244075). Fourexperiments (designated A, B, C, and D respectively) were performed.Each experiment compared embryos obtained by manual excision to embryosobtained by a method of the present invention: excision by a liquid jet(experiments A, B, and D) or excision by a gas jet (experiment C). Theliquid jet in experiments A and B used ordinary tap water and a nozzlemade of a pipette tip. Experiment C tested a gas jet using air from acompressed-air pump and a nozzle made of a pipette tip. The liquid jetin experiment D used ½ strength MSPL medium (see Table 1 of United StatePatent Application Publication Number 2004/0244075 to Cai et al.) as theliquid and a solid stream nozzle with an equivalent orifice diameter of0.020 inches (catalogue number TP000050-SS, Agricultural Division ofSpraying Systems Co., Wheaton, Ill.).

Corn ears were harvested twelve days after pollination and sterilized bysoaking in a 1-liter bottle of 80% ethanol for 3 minutes. Embryos weremanually excised by cutting off the top third of the kernel with ascalpel and removing the embryo from the kernel using a narrow spatula.The collected embryos were excised into 1 milliliter of ½ MSPL medium ina single microcentrifuge (Eppendorf) tube. The medium was removed andreplaced with 1 milliliter of Agrobacterium tumefaciens prepared asdescribed below

Embryos were also substantially isolated using a fluid (liquid or gas)jet, following procedures similar to those described in Examples 10 and11. The fluid jet was used to excise the remaining embryos on the earsafter removing the top third of the kernel with a scalpel. The ear waspositioned so that the fluid jet was aimed into individual cut kernelsin succession to dislodge both the embryo and non-embryo tissue(endosperm). The kernel contents removed from the ear were passedthrough a coarse screen to remove large pieces of endosperm, and theembryos were collected on sterile cheesecloth. Embryos were transferredusing a small spatula from the cheesecloth into a microcentrifuge tubecontaining 1 milliliter of ½ MSPL medium. After all of the embryos werecollected, ½ MSPL medium was removed and replaced by 1 milliliter ofAgrobacterium tumefaciens inoculant.

Embryos prepared by the various excision methods were subjected to thesame inoculation, selection, and regeneration procedures. Suitableprocedures, including descriptions of media and reagents, fortransformation of plants using glyphosate selection and GFP as areporter have been disclosed in United State Patent ApplicationPublication Number 2004/0244075 to Cai et al.

Embryos were inoculated with 1.0 milliliters of Agrobacterium for 5minutes. The contents of the microcentrifuge tube were poured onto aplate of co-culture medium, excess Agrobacterium was removed by pipette,and the contents were co-cultured for 18 hours at 23 degrees Celsius.Embryos were transferred next to induction MS medium, and cultured at 30degrees Celsius for 13 days. Calli derived from the transformation werecultured at 27 degrees Celsius for 11 days prior to regeneration. Atthis time, GFP positive sectors were counted using a fluorescencemicroscope. For regeneration, calli derived from each embryo wereindividually transferred to MS/6 BA medium and cultured in a light roomfor 7 days, after which each greening callus was transferred to MSODmedium and returned to the light room for 17 additional days. Resultingshoots were transferred to Phytatrays™ containing regeneration medium(consisting of 2.165 g MS basal salts, 5 milliliters 100× MS vitamins,and 20 grams sucrose made up to 1 liter in water and autoclaved, pHadjusted with KOH to 5.8, solidified by autoclaving with 3 g Phytagel™,and with 0.75 milliliters of 1 milligram per milliliter indole-3-butyricacid, 0.5 milliliters of 1 milligram per milliliter 1-naphthaleneaceticacid, and 0.2 milliliters 0.5 molar glyphosate added). After about 3weeks, transgenic plants were hardened off by transplanting rootedshoots in peat pots containing soil mix and grown at 26 degrees Celsius.

The results of these experiments are summarized in Table 3. The numberof embryos that were transformable is estimated from the number ofGFP-positive embryos.

TABLE 3 Viability and transformation frequency of excised embryos.number of number of number of transformation/ excision embryosGFP-positive transformation plants regeneration experiment methodinoculated embryos frequency to soil frequency A manual 56 23 41% 6 11%liquid jet 44 8 18% 3 6.8%  B manual 22 11 50% 6 27% liquid jet 23 4 17%1  4% C manual 33 27 82% n/a n/a gas jet 61 19 31% n/a n/a D manual 3617 47% n/a n/a liquid jet 166 51 31% n/a n/a n/a: data not available

Overall transformation and regeneration frequency is given as thepercentage of GFP-positive plants regenerated from the inoculatedembryos. These results demonstrate that various methods and devices ofthe present invention are useful for providing multiple monocot embryossuitable for genetic transformation or tissue culture.

Example 15. Development of Liquid Media for Excising Corn Embryos

The fluid jet apparatus required about 20 L of liquid for excisingembryos from one ear, necessitating the development of an excisionmedium that is simple to prepare (i.e., contains only about one or twoingredients), can be easily prepared, is filter sterilizable preferablythrough an in line filtration unit, can flow through the fluid jetapparatus at a preferred operating pressure of 40-60 psi, and does notrequire adjustment in pH prior to use. Such media and its preparationwould lower cost, reduce media preparation time, and allow automation.This example provides several such media.

Culture media such as Lynx 1013 (inoculation medium) and Lynx 1902 (halfstrength Lynx 1013) were successfully used for excising corn embryosuseful for transformation using a fluid jet apparatus. The Lynx 1013comprises (per liter): MS Basal Salts (Phytotech; PhytoTechnologyLaboratories, Shawnee Mission, Kans.): 2.165 g; MS Vitamins (100×;Phytotech): 10 ml; Glucose (Phytotech): 36 g; Sucrose (Phytotech): 68.5g; Proline (Fisher): 0.115 g. The medium was adjusted to pH 5.4 with KOHthen filter sterilized. Although these media worked well, they containeda number of ingredients, some of which must be filter sterilized. Thesemedia also require pH adjustment prior to use.

Several other liquid media (Table 4) were tested for excisingtransformable corn embryos from corn ears. Excised embryos were thenused for transformation according to methods described elsewhere in thedescription, and the transformation frequency (TF) was determined foreach tested excision medium. TF was defined as the number of uniquetransformation events regenerated into plants divided by the number ofembryos inoculated with Agrobacterium.

All media tested, including water, were able to produce corn embryosthat were transformable (see Table 4). However, media comprisingmannitol produced comparable TF to control medium Lynx 1902. Themannitol medium is a simple medium with only two ingredients (mannitoland water), does not require pH adjustment prior to use, and is filtersterilizable, making it significantly more cost effective and convenientto use.

The mannitol concentration in the medium is from about 0.05 M to about0.5M. Preferably, the mannitol concentration in the medium is about 0.1M. Most preferably, the mannitol concentration in the medium is about0.2 M. A suitable concentration of mannitol for the excision medium,however, can be determined by those skilled in the art of plant tissueculture, by simply varying the mannitol concentration.

Representative osmotic potential measurements of selected media arefound in Table 4. Readings were taken using Wecor 5100C Vapor PressureOsmometer (Wecor, Logan, Utah, USA), with calibration using standardsolutions of 100, 290, and 1000 mOsm/kg. A medium with an osmoticpotential (i.e. molality) from about 0 mOsm/kg to about 500 mOsm/kg issuitable for excising corn embryos for tissue culture, including forinstance about 7 mOsm/kg to about 300 mOsm/kg. For instance, a 0.2 Msolution of mannitol in water has an osmolality of about 222-230mOsm/kg, while a solution of 0.05% MES in water has an osmolality ofabout 7 mOsm/kg. This range of osmotic potential is preferably obtainedby adding one or two compounds to make the medium, such as Lynx media#1937, #1932, and #1162. Preferably, such compounds have no significantadverse effect on tissue culturability and transformability of theexcised embryos.

TABLE 4 Osmotic potential measurements of exemplary solutions for use inisolating embryos. Mean Osmolality STD Identifier and compositionmOsm/kg Osmolality % TF Lynx # 1118 - Sterile distilled water 7.00 8.893.0 Lynx # 1013 - Inoculation Medium 503.67 9.07 20.6 Calcium chloride-10 ppm CaCl₂ n/a n/a 4.2 Lynx # 1902 - Half Strength 262.67 4.04 9.5Lynx # 1013 Lynx # 1932 - 0.2M Mannitol, 222.33 9.29 15.6 autoclavedLynx # 1986 - 0.2M Mannitol, 230.00 28.83 11 filter sterilized Lynx #1987 - 0.1M Mannitol, 117.33 27.15 9.7 filter sterilized Lynx # 1162 -5% Sucrose (w/v) 258.33 4.51 4.5 Lynx # 1953 - 5% Sucrose + 313.33 3.511.6 MS Salts (same amount as in Lynx #1013) Lynx # 1937 - 0.05% MES, pH5.4, 7.00 3.61 0.3 autoclaved Lynx # 1964 - 0.05% MES, pH 5.8, 13.3311.50 0 filter sterilized with 0.2 micron filter n/a = not done

Example 16: Construction of a Media Preparation System for Fluid JetExcision

This example describes a media preparation system (MPS) for use with afluid jet apparatus. The fluid jet apparatuses tested required about 20liters of medium, which requires time and effort to make and use. TheMPS of the present invention can prepare large amount of media quickly.It also allows for the preparation of a specified volume of media for aparticular size corn ear.

The MPS comprised a Mixing Chamber (MC) and an MPS Housing (MPSH). TheMC is designed to be easily disengage able from the MPSH for easysterilization by autoclaving. Both the MC and MPSH can be made of anysuitable material. Preferably, they are made of a material such as steelor aluminum. As shown in FIGS. 11 and 12 , the MC comprises a tankhaving an upper end and a lower end, a flange that is attached to theupper end of the tank, and a cover plate for covering the upper end andfor sealing the mixing chamber. Preferably the tank is sealed with thecover plate by an o-ring provided in the flange. The cover plate ispreferably made of 7075 aircraft grade, corrosion resistant aluminum toreduce weight of the mixing chamber. In one embodiment, the tank isprovided with three pins at the lower end, each on three sides as shownin FIG. 12 , for orienting and positioning the tank on the mixingchamber supporting plate. In a working position, the three pins engagewith the three positioning brackets provided on the supporting plate asshown in FIGS. 12 and 14 . A positioning plate (FIG. 12, 13 ) is alsoprovided at the bottom of the tank, at the forth side, for holding thetank in a working position on the mixing chamber supporting plate.Preferably, the positioning plate has a ¼″ NPT thread for mounting aquick connector for carrying a position arm with a handle (FIG. 14 ) inorder to fix the mixing chamber on the supporting plate (e.g. FIG. 13 ).

The cover plate is provided with an opening for inserting a tube forbringing in a liquid such as water for making medium, an opening forinserting a tube for taking out the prepared medium which is connectablea fluid jet apparatus, an opening for inserting a tube for adding mediumingredients, preferably of stainless steel, into the mixing chamber(e.g. FIG. 12 ). In the working position, separate o-rings seal varioustubes with openings. The cover plate is also provided with an openingfor inserting a housing containing an ultrasonic sensor for sensing aspecified volume of medium that need to be prepared. Preferably, theopening for the ultrasonic sensor housing is provided on one side of thecover plate. In the working position, various openings provided on thecover plate of the mixing chamber are connected to the correspondingtubes provided at the lower surface of the upper platform of the housing(FIG. 11 ). These tubes are preferably made of steel. The variousopenings connect to the mixing chamber as shown in FIG. 11 , forinstance via connecting tubing. The tubing is preferably made ofpolycarbonate.

A media mixer assembly is attached to the lower end of the cover platesuch that the assembly hangs, preferably in the centre of the tank. Theassembly comprises a two blade folding impeller, a stainless steel shaftsealed by an o-ring in the cover plate, a high temperature ball bearingsuitable for functioning at a high temperature, such as above 400° F.,present during autoclaving and a spider coupler (e.g. FIG. 12 ) forcoupling the assembly with a motor in the MPSH (FIG. 16 ).

A ball nut-mounting block is mounted on the lower side of the mixingchamber supporting plate and is connectable to a ball screw mounted in aball bearing provided at the upper surface of the lower platform of thehousing. The support plate is also slidably connected via four wheels tofour legs of the MPS housing as shown in FIG. 15 . The wheels roll alongin t-slots of the four legs to facilitate up and down movement of themixing chamber when the stepper motor provided at the lower surface ofthe lower platform of the MPSH is turned on.

After autoclaving, the mixing chamber is positioned on the supportingplate by the three pins and the positioning plate. The positioning armwith the handle is inserted in the quick connector. The arm is pusheddown and turned 90 degrees clockwise with the help of the handle forpositioning and fixing the mixing chamber on the supporting plate. Thepositioning arm is attached to a cam and mounted on a spring that iswelded to the supporting plate. The spring pushes the arm and the camup. When placed in position, the back pin of the mixing chamber pushesan electrical switch, which sends a signal to a Programmable LogicController (PLC). This signal informs the PLC that the mixing chamber ispositioned on the mixing chamber supporting plate. The stepper motorprovided on the lower platform of the housing then pushes the mixingchamber towards the upper platform of the housing where, in a workingposition, various openings provided on the cover plate of the mixingchamber become connected to the corresponding tubes provided at thelower surface of the upper platform of the housing (FIG. 16 ).

Upon receiving an operator signal, the PLC opens an electromagneticvalve connected to a water system for filling the mixing chamber withwater. The ultrasonic sensor monitors the water level in the mixingchamber. When the level of water reaches the specified level in themixing chamber, the PLC closes the water electromagnetic valve and waitsfor a signal from the operator indicating that the components of theexcision medium are in the mixing chamber. When the PLC receives thatsignal, it actuates a motor provided on the upper surface of the upperplatform of the MPSH and operably linked to the mixer assembly. Thus,the mixer assembly starts mixing the medium. Once the medium is ready,it is pumped using a gear pump through a medium outlet tube to the fluidjet apparatus according to a PLC program.

In one embodiment, the prepared medium outlet/supply connected to thefluid jet apparatus is provided with an inline filtration unit such as a0.2 Micron Absolute FiberFlo® Hollow Fiber Capsule Filter (MinntechFiltration Technologies Group, Minneapolis, Minn.) for sterilizing themedium before its use in the fluid jet apparatus for excising embryos.

Example 17: Embryo Isolation by Phased Excision

This example describes an apparatus for preparing multiple corn embryossuitable for tissue culture from kernels on a corn ear, comprising atleast one aperture for guiding a first fluid stream at a first timepoint for substantially extracting endosperms from the kernels, and asecond fluid stream at a second time point for substantially extractingembryos from the kernels, the extracted embryos being suitable fortissue culture. The apparatus further comprises a means for moving atleast one corn ear relative to the first and the second fluid streams.The means for moving at least one corn ear relative to the first and thesecond fluid streams rotates at least one corn ear and the at least oneaperture relative to each other. The means for moving at least one cornear relative to the first and the second fluid streams moves the fluidstreams along the longitudinal axis of at least one corn ear. In thisparticular embodiment of the apparatus, each fluid stream is a liquidstream. The liquid stream may consist, for instance, essentially ofmannitol and water at a concentration of about 0.05 M to about 0.5 Mmannitol. The fluid stream can alternatively be a gas stream. Theapparatus may further comprise a means for detecting excised endospermsor embryos. The apparatus may also further comprise a means forchanneling excised endosperms or embryos so as to separate endospermfrom embryo. The apparatus further comprising a means for linking themeans for detecting excised endosperms and embryos to the means forchanneling excised endosperms or embryos electronically, for automation.The apparatus can further comprise at least one separator for separatingembryos from non-embryo tissues, wherein the separated embryos comprisecorn embryos suitable for tissue culture. The separator can comprise asize-exclusion device. Alternatively, the separator can separate theembryos from the non-embryo tissues by differential hydrophobicity or bydensity differential.

The present example also describes a method of providing monocot embryossuitable for tissue culture comprising: (a) providing monocot seedscontaining embryos having an opening in the pericarp of the seeds; and(b) applying a first force at a first time point to the seeds forsubstantially extracting endosperms from the seeds and a second force ata second time point for substantially extracting embryos from the seeds,the extracted embryos being suitable for tissue culture. The forcescomprise one or more forces selected from fluid jet positive pressure,liquid jet positive pressure, mechanical positive pressure, negativepressure, centrifugal force, linear acceleration, linear deceleration,fluid shear, fluid turbulent flow, and fluid laminar flow. The methodfurther comprises separating embryos from non-embryo tissue by themethods selected from the group consisting of size-exclusion,hydrophobic separation, and density differential separation. The monocotseeds are provided on at least one ear. The monocot may be in the familyPoaceae, such as a Zea species (e.g. Zea mays). The step of tissueculture may include one or more steps of transformation andregeneration, resulting in at least one fertile transformed plant.

As shown in FIG. 17 , the apparatus is provided with a flat jet nozzle(1) for producing a flat laminar flow (6), two stepper motors controlledby a programmable logic controller (PLC), and a digital gear pump. Onestepper motor rotates the ear (2), having corn kernels from which crownhave been removed to facilitate excision, mounted on a shaft in ahermetic chamber (3) and the second motor advances the ear along theaxis such that the flow acts on each kernel for substantially the sameamount of time. The digital gear pump forces the liquid medium throughthe nozzle as specified by the PLC rate and pressure. The nozzle (1)produces a flow of liquid that is about 0.003″ wide and about 1″ high.Generally, the width of the flow is less than the width of the kernel.In the tested pressure range 30-75 PSI, the laminar flow was stable at adistance up to 2.5-3″ from the nozzle (1) and did not fall apart beforereaching the ear. The ear (2) may be positioned 1¾″-2″ away from thenozzle (1) so that the flow of liquid acts on each kernel in a row withsubstantially the same force. The amount and duration of force can bemanipulated by altering the pressure of liquid in the nozzle, byadjusting the distance of the ear to the nozzle, and the space betweenthe nozzle plates as well as other parameters such as the entry angle ofthe flow of liquid into the kernel, and the speed of ear rotation.

In the first phase, a PLC according to a PLC program opens a channel (4)by valve (5) for endosperm collection, and sets a pressure and flow ratethrough the nozzle (1) by sending control signals to the digital gearpump, and rotates and advances the ear (2) by the two stepper motors.The flow of liquid (6) controlled by the PLC first washes out thekernel's (7) endosperm (8) leaving embryos (9) attached to the kernels(7). The amount of time and force necessary to wash out the endospermmay be adjusted depending upon the endosperm size, extent of cut in thecrown of the kernel, and differences between ear and kernel sizes, amongother parameters. The mixture of liquid medium and endosperms collectedin channel (4) can be pumped through a filtration system. Afterfiltration, the liquid medium can be reused in the excision process.

In the second phase, the PLC according to the PLC program closes channel(4) and opens a channel (10) by valve (5) for embryo collection, settinga pressure and flow rate through the nozzle (1), and rotating andadvancing the ear. The flow of the liquid (6) washes out embryos (9) andthe rest of endosperms (8) out of the kernels (7). Further separation ofembryos, if desired, can be done using separators described elsewhere.The amount of time and force necessary to wash out the embryo can beadjusted depending upon the embryo size, extent of cut in the crown ofthe kernel, and differences between ear and kernel sizes, among otherparameters. The mixture of liquid medium and embryos collected inchannel (10) is subjected to a separation device (e.g. Example 18) toisolate embryos from debris and liquid, and the liquid can be pumpedthrough a filtration system for reuse in the excision process.

The chamber (3) may optionally be provided with an emitter (11) andthrough beam sensor (12) for detecting the embryos in exiting liquidmedium after excision. During the first phase, the signal confirmingappearance of embryos in the exiting media instructs the PLC forcompleting phase one. During the second phase, the signal confirmingdisappearance of embryos in the exiting liquid medium alerts the PLC forcompleting phase two, and may further instruct the PLC to shut down theapparatus.

Corn embryos excised using this apparatus and method were transformedusing standard methods known in the art of corn transformation. (e.g.Cal et al; U.S. Patent Application Publication 2004/0244075). Alltreatments used Lynx # 1986 (0.2M Mannitol, un-sterilized), for excisingembryos. Representative transformation frequencies (TF) are shown inTable 5, indicating that embryos produced by this apparatus and methodwere transformable and yielded transgenic plants.

TABLE 5 Transformation of corn embryos excised by the phased fluid jetapparatus and method. Exp. # Trt # Experiment Description TF % 6778 2 FJPhase w/endosperm 6.0% 6778 3 FJ Phase w/o endosperm 8.0% 6858 2 FJPhase w/endosperm 4.0% 6858 3 FJ Phase w/o endosperm 8.0% 6858 5 FJPhase w/endosperm 2.9% 6858 6 FJ Phase w/o endosperm 5.7% 6894 2 FJPhase w/endosperm 14.0% 6894 3 FJ Phase w/o endosperm 16.0% 6894 5 FJPhase w/endosperm 6.7% 6894 6 FJ Phase w/o endosperm 6.7% “FJ (FluidJet) Phase w/endosperm” refers to embryos that, after collection, wereinoculated with endosperm and left on the same plate (embryos andendosperm together). “FJ Phase w/o endosperm” refers to embryos that,after collection, were moved to a fresh plate of media leaving theendosperm behind.

Example 18: Embryo Separation Process

This example describes a flotation process for separating embryos from amixture produced by a fluid jet excision process. Parameters forefficient separation are described herein. In this process, bubbles of agas were generated in a fluid and attached to excised corn embryospresent in the fluid. The bubbles floated to the surface (i.e. fluid-airinterface), allowing the embryos to be preferentially collected whileleaving behind endosperm material and other debris.

To reduce the number of bubbles per unit volume and generate a moreuniform field of bubbles so that less shear force was generated, adevice was constructed containing multiple point sources of bubbles. Thedevice was constructed by inserting individual splinters of limewoodinto pre-punched holes in a length of silicone tubing (Masterflex®silicone tubing C-96400-16, Cole-Parmer). After inserting the limewoodslivers into silicone tubing, one end of the tubing is plugged with astainless steel bearing. The tubing is then coiled in a spiral andsecured with a metal wire as shown in FIG. 18 . The spiral-shapedlimewood air dispersion device combined with using PEG as the surfactanthas shown 95% embryo purification from nearly all of the endosperm asshown in FIG. 19 . FIG. 20 illustrates the amount of endosperm separatedfrom the embryo fraction by bubble flotation using the spiral shapedlimewood air dispersion device and PEG. Other limewood-based bubblersmay be constructed or purchased from, for instance, aquarium suppliers.The Petri plate on the left in FIG. 20 shows the large amount ofendosperms intermixed with embryos when the flotation process was notused. In comparison, the Petri plate on the right shows the reduction inthe amount of endosperm co-fractionating with the embryos when thefloatation process of the present invention was used. Bubbles can alsobe produced by ceramic materials having various pore sizes.

In order to detach bubbles from the source material generating them, itis desirable that the source material be of an opposite nature to thebubbles, with respect to the degree of polarity (i.e. hydrophobicity).Since the air bubbles are covalent in nature, they will tend to detachreadily from a material that is polar in nature. Conversely, bubblesemerging from a surface which is also covalent in nature, like a porousplastic sparger, will tend to adhere to the surface, and coalesce andgrow to a larger size before detaching. In this respect, the Chemglasssparger, limewood, and ceramic are all desirable choices. The surface oflimewood is composed of cellulose, hemicellulose and lignin which arepolar and thus hydrophilic, as are the sintered glass sparger andceramic materials

In one instance, once embryos were carried to the surface by the bubblesand deposited in the froth, they were harvested by skimming off thefroth and then rinsed free of the froth. In another experiment, a vacuumfilter device (e.g. FIG. 21 ) was used to harvest embryos off thesurface of the froth.

In one experiment, embryos and endosperm were excised from donor earsusing the fluid jet (FJ) apparatus and collected on cheesecloth (CC).The mixture of embryos and endosperm was then placed in a graduatedcylinder filled with Lynx 1013 and 20 μl of 20% PPG (polypropyleneglycol mono-butyl ether—Av. Mol. Wt. 340; Aldrich Cat. No. 438103; SigmaChemical Co., St. Louis, Mo.), and bubbles were produced by air pumpedthrough a fitted fine pore glass dispersion tube (Chemglass) by anaquarium pump (bubbler). Separated embryos, substantially free ofendosperm material and other debris, were collected as outlined above.Separated embryos were used for Agrobacterium-mediated transformation ofcorn by using standard methods known in the art of corn transformationas noted above. Transformation frequencies are shown in Table 6.

TABLE 6 Transformation of corn embryos separated by flotation using airbubbles stabilized by PPG. # # Exp. Trt Experiment embryos # Events toTF # # Description inoculated GFP+ Soil % 6376 2 FJ Bubbler CC 120 32 97.5% 6396 2 FJ Bubbler CC 223 32 10 4.5% 6419 2 FJ Bubbler CC 201 87 178.5%

In another experiment, a limewood-based bubbler was used to separateembryos from endosperms excised by the fluid jet apparatus. In generalthe limewood-based bubbler was placed in a beaker in a sterile laminarflow hood. The tubing of the bubbler was connected to an air filter atone end. The other end of the air filter was connected to the aquariumpump. The separation medium (0.2M Mannitol with 20 μl of 20% PEG (Avg.Mol Wt. 8000, Sigma No. P21390)) was then added to the beaker over thebubbler. The embryo and endosperm from the fluid jet apparatus werecollected and transferred to the beaker. The air was bubbled through thebubbler. Floating embryos were transferred to a small Petri dish andused for transformation using standard methods known in the art of corntransformation as noted above. Transformation frequencies are shown inTable 7.

TABLE 7 Transformation of corn embryos separated by flotation using airbubbles stabilized by PEG Experiment # of embryo used Mean TF (%) SE (%)Manual Excision 1759 22.33 4.58 Fluid Jet Excision 1700 34.85 5.62 w/oseparation Fluid Jet Excision 1648 35.50 5.70 with Bubble Separation

Example 19: Apparatus for Separating Embryos by Flotation

FIG. 22 shows an apparatus for separating embryos by flotation thatcomprises a floatation column (E) connected to an inlet tube (A) at theupper end of the column for adding a mixture of embryos, endosperm andother debris, an air dispersion tube (G) at the lower end of the columnfor producing air bubbles for raising the embryos, and an outlet tube(H) also at the lower end of the column for regulating the liquid leveland for removing the debris from the bottom of the column. The bottom ofthe column is preferably slanted to facilitate removal of the debristhat does not float. The floatation column is further provided with anoverflow chute (C) for collecting the froth containing the embryos. Theair dispersion tube is further provided with a valve (F) to preventbackflow of column liquid into the air dispersion tube.

To operate this apparatus, the flotation column is filled with a liquidmedium compatible with subsequent transformation and tissue culturing ofthe embryos until the level of the liquid reaches the overflow drain(D). The overflow drain has an anti-siphoning tube (B) open to the airat the top. A surfactant is added to the liquid medium to prevent thepremature coalescence of the bubbles and stabilize the froth forattaching and raising the embryos to the top of the flotation column.

The next step in operation of the apparatus is to turn on the air supplyto the air dispersion tube. The pores in the dispersion tube are smallenough to produce bubbles that rise slowly enough that their contacttime with the embryo's hydrophobic surface is long enough to allowattachment of the bubbles to the embryos.

Next a mixture of embryos, endosperm, and debris produced by the fluidjet apparatus containing the desired concentration of a surfactant (suchas PEG at a concentration of 20 parts per million) is fed into the inlettube. As the slurry enters the flotation column the suspended particlesencounter the column of bubbles from the air dispersion device. Bubblesfirst attach to the embryos and preferentially raise the embryos to thesurface of the flotation column where they become part of the froth. Asthe froth accumulates it exits the flotation column through the chute(C).

During operation the column can be tilted at a slight angle fromvertical toward the chute so that the rising bubbles are concentrated atthe back wall of the column and tend to push the froth forward and outthe chute. Alternatively, a cover which slants at an upward angle towardthe chute can direct the froth to the chute.

The endosperm-rich debris which does not float falls to the bottom ofthe flotation column and accumulates at the low point of the slantedbottom. These debris are displaced out of the column during theautomatic liquid leveling of the column that occurs when the slurry ofembryos and endosperm debris is initially fed into the feed funnel atthe beginning of column operation, or may be removed at the end ofoperation of the flotation column.

In another embodiment, the level of liquid in the flotation tank is keptlevel with an exit chute and the froth rising to the surface iscontinuously swept into the chute. This action can be facilitated byhaving more bubbles rising on the side of the tank away form the chuteso that there is a circulation produced which rises on the side awayform the chute and sinks on the side with the chute.

In another embodiment, the liquid level in the flotation tank can bekept below the level of the chute until a sufficient quantity of frothand embryos have accumulated and then the liquid level is raised so thatthe froth and embryos spill into the chute.

Example 20: Alternative Apparatus for Separating Embryos by Flotation

This example describes another embodiment of the apparatus designed toseparate corn embryos from corn endosperm by means of a flotationprocess using bubbles and surfactants. The apparatus can be used with anembryo excision apparatus such as a fluid jet apparatus describedelsewhere in the application.

In this apparatus (FIG. 23 ), the embryos and endosperms flow into achamber provided with a means for producing bubbles. The bubbles can begenerated by forced air through a sparger. Preferably the bubble size(diameter) is about 100 μm to 500 μm. A bubble stabilizing agent such aspolyethylene glycol (PEG) is also added at a concentration of about 1 toabout 100 ppm, for instance 20 ppm. The embryos preferentially attach tothe bubbles through hydrophobic interaction and the bubbles raise theattached embryos to the surface of the chamber. Addition of PEG enablesthe bubbles to carry their “load” of embryos to the surface. The PEGalso helps to create a froth layer at the top of the surface and embryoscollect on top of the froth layer where they are carried into theoverflow outlet. A motorized skimmer device can be used to furtherfacilitate flow of the froth containing embryos into the outlet wherethey pass through an imaging station or a counting device for countingthe number of embryos and collected in containers as needed. A switcharm operably linked to the imaging station or the counting device can beprovided to dispense desired numbers of embryos into differentcontainers. The containers can be connected to a vacuum manifold deviceto remove the liquid leaving behind the embryos for further use.

The apparatus is further provided with a means for bringing liquid inand out of the chamber. The liquid level in the chamber can be managedby a liquid leveling device. Endosperms and other debris that are notselectively attached to the bubbles are discarded through the means fortaking liquid out at the bottom of the container, such as a wasteoutflow drain. The same liquid medium that is used for excising theembryos can be used to fill the chamber for separating the embryos. Themedium coming out of this apparatus can be recycled for further useduring excision and separation processes.

Example 21: Combination Device for Excising and Separating Corn Embryosfor Tissue Culture

This example illustrates a combination device or an integrated devicecomprising an embryo extractor as shown in FIGS. 24-25 and an embryoseparator as shown in FIG. 26 . Preferably, the embryo extractor is incommunication with the separator. However, the embryo extractor or theseparator can be operated separately and the output of the embryoextractor can be the input of the embryo separator.

Referring to FIG. 24 which is a top view, the embryo extractor isprovided with at least one fluid jet (C) for extracting embryos fromeach cob (B). More than one fluid jet may be directed at a single cob.Each cob in FIG. 24 is held in place by spring-loaded retainers (A) atone end of the cobs and a powered retainer (D) at the other end whichmay be driven by a belt mechanism (E). A side view of the apparatus isshown in FIG. 25 . Normally, the crowns of the kernels on the cob arepartially or fully removed to facilitate embryo extraction.

Any liquid medium can be used to produce the jet flow (C), for instanceas described in Example 15. Sterile water can be used to excise embryossince the time between initial embryo contact with water and immersionin the culture medium in the flotation tank is short. A filter can beused to sterilize the water. Alternatively, the liquid medium comprisesmannitol or other solute of suitable osmotic strength as describedabove.

As shown in FIG. 26 , after extraction of the embryos from kernels, theembryos and debris optionally fall onto, or are added onto, an embryoseparator comprising a double screen conveyer belt system connected to aflotation chamber. The double screen conveyer belt has a moving coarsescreen (A) which catches coarse material, but allows embryos and otherfine material to pass through to a fine screen (H) which retains theembryos and similar size material, but allows very fine material andliquid to pass through to the waste liquid chute (W). The materialcollected on the coarse screen is washed loose in the coarse screen washtank (F) and is aided by the flow of waste down the waste liquid chute(W) and out a drain through the coarse screen wash tank overflow (E) andwash tank drain (G). The material collected on the fine screen (H) iswashed loose in the fine screen wash tank (N) with the help of freshmedium and preferably a frother through inlet (M). If embryos do notdislodge from the fine screen (H) into the fine screen wash tank (N),agitation can be used. For example, embryos captured on the fine screenare disloged into the floatation medium as they pass around roller (K)and also by impact of make-up medium added via (M). The delivery offresh media and the frother is adjusted so that debris accumulating inthe floatation chamber are continually removed through the liquid levelregulator inlet (U) and outlet tubes (V).

Molded thermoplastic meshes, which are not woven, may be preferable touse in this apparatus since they do not unravel. Such meshes areavailable (e.g. McMaster-Carr, Atlanta, Ga.) in various screen (strand)thicknesses, strand widths and mesh opening sizes. Meshes made ofpolypropylene are also preferable because they can be autoclaved. Theprecise mesh dimensions may be empirically selected by the operatordepending on the expected sizes of embryos and debris that areintroduced to the separator.

Referring to FIG. 26 , the coarse screen (A) and the fine screen (H) canbe supported by rollers. Preferably, the coarse screen (A) is supportedby a powered roller (B), an idler roller (C), and a wash roller (D). Thefine screen (H) is supported by a powered roller (I), idler roller (J),a wash roller (K), and a lifter roller (L). The rollers may be straightcylindrical, or other than straight cylindrical, preferablyconcave-cylindrical since these allow troughed belts to be used whichtypically have less loss of product along their edges than flat belts.Troughed belts may allow higher belt speeds and inclines. One or more ofthe rollers may be powered, for example roller (B), and may have theirsurfaces textured to more firmly grasp the screen. It may be desirableto power only one roller directly and have the other powered, forexample roller (C), from the first as with a belt or gear. Lateral andcentral supports or guides under the coarse and fine screens may bedesirable to prevent sagging and loss of liquid along the edges of thescreens. Additional support rollers may also be desirable.

After the material collected on the fine screen (H) dislodges in thefine screen wash tank (N) it falls past the bubble deflector (O) andenters the flotation chamber (P) where it encounters rising bubbles ofair (S) produced by, for example, by an air dispersion tube (T). At thispoint bubbles of air attach preferentially to the hydrophobic surfacesof the embryos, carrying them to the surface where they are deposited ina froth through a flotation chamber side opening (Q) and embryo delivery(froth) chute (R). If the embryos descending in the flotation chamber(P) are not exposed adequately to the bubbles, a mixing device such as amagnetic stirrer can be provided in the lower portion of the chamber (P)to improve exposure.

The liquid level in the flotation tank is regulated by the enteringfresh medium and the frother from the inlet (M) and by the liquidleveling system (e.g. FIG. 27 ) comprising an inlet tube U and andoutlet tube (V) in which excess liquid along with debris enters the tubeU and exits the tube (V). A U-shaped tube connects (U) and (V) with thehighest level in the tube reaching the level at which the liquid is tobe maintained. The top of the U-tube is also provided with ananti-siphoning opening. Other liquid leveling device such as thosefloat-based, optics-based, conductivity-based, and electric-based can beenvisioned to regulate the level of liquid in this device.

Example 22: Apparatus for Isolating Transformable Tissues from Seeds andFruits

The compositions, methods, and apparatuses of the present invention canbe used to isolate transformable tissue, for example, embryos from seedsand fruits of other monocot and dicot plants, including, withoutlimitation, corn, wheat, barley, soybean, sunflower, cotton, canola,peppers, tomatoes, raspberry, and strawberry, among others.

A suitable holder, for example, a sheet having holes and/or slotssuitable for holding seeds or fruits of various shapes and sizes isprovided (e.g. FIG. 28 ). The holder can be made from a suitablematerial such as plastic or metal. The holder can be a flat sheet orrolled into a cylinder as shown in FIG. 29 , and suspended in a gaseousphase such as an air or liquid phase or may be partially suspended inthe gaseous and the liquid phases. The seeds and fruits can be held ontothe holder by a suitable force, such as a mechanical and/or suctionforce. The holder can be fixed relative to a fluid force, for example aliquid stream of the medium described in Example 15 or movable relativeto the fluid force.

In another embodiment shown in FIG. 30 , a pressure Cam or Screw, forexample an auger, is inserted at one end of the cylinder to applypressure on the seeds and fruits to further facilitate the isolation ofembryos. The isolated embryos can be separated from debris by severalmethods described elsewhere in the specification.

In another embodiment of the present invention, the sheet or cylindermay be centrifuged in a container to apply forces on the retained seedsor fruits to isolate embryos.

The embryos can be separated from other tissues as described in Examples18-20 and used for transforming and regenerating various plant species.For instance, soybean embryos can be transformed using methods describedin U.S. Pat. No. 7,002,058. Transformation methods for other plants areknown in the art.

Example 23: Methods for Separating Cotton and Soybean Embryos

This example illustrates the utility of compositions, methods, andapparatuses of the present invention in separating cotton and soybeanembryos from seed tissue, thus demonstrating wider utility. Cotton andsoybean seeds were crushed by the method and apparatus described in U.S.patent application Ser. No. 12/045,502, filed Mar. 10, 2008, and U.S.Patent Application Publication 20050005321 and added to a solution of0.2M Mannitol with 0.0012% PEG 8000 for isolation of cotton embryos andabout 0.003% PEG 8000 for isolation of soybean embryos, using theapparatus shown in FIG. 31 . Bubbles were produced for instance asdescribed in Example 18. Most of the cotton embryonic axes were found toaccumulate with the bubbles as shown in FIGS. 32-33 . The cotton embryoswere prepared for transformation using the methods described in U.S.patent application Ser. No. 12/045,502. In the case of soybean, thebroken embryos (embryonic axis and cotyledons) were found to be at thebottom of the container while most of the seed coats floated to the topwith the bubbles. The embryonic axes and cotyledons were then furtherseparated by density differential methods. For example, in a 6.5% Ficollsolution in 20% sucrose embryonic axes rose to the surface whilecotyledons sank to the bottom of the container. The soybean embryos canbe transformed using the method described in U.S. Pat. No. 7,002,058.

Example 24: Development of a Co-Culture Medium for EnhancingTransformation Frequency

In some experiments, use of co-culture medium 1233 (U.S. Patent Applic.Publn. 2004/00244075) in the corn transformation process, utilizingembryos prepared by a fluid jet method and separated by a flotationmethod, resulted in lower transformation frequencies (TFs). In order tomaintain or enhance transformation frequency, a new co-culture medium,termed “1898,” was tested and unexpectedly found to enhance TFs.

Several set of experiments were conducted to compare co-culture medium1233 and co-culture medium 1898. Among other compositional differences(see Table 9), co-culture medium 1898 has a lower level of 2,4-D and hasCarbenicillin. Immature embryos from corn ears were excised andseparated as described in Examples above. Separated embryos were splitinto two treatments. Embryos in both treatments were inoculated withplant transformation vector pMON97367 comprising a gus and a CP4expression cassette. The embryos for treatment 1 were co-cultured onmedium 1233 and the embryos for treatment 2 were co-cultured on themedium 1898. After overnight co-culture, embryos were processed by themethod described in the US Patent Application Publication 2004/00244075.

Table 8 shows that the use of co-culture medium 1898 resulted inimprovements in all key performance indicators such as culture response,events created, events transferred to phytatrays, events transferred tosoil, and % TF in comparison to the use of the co-culture medium 1233.Table 9 gives compositions of media used.

TABLE 8 Enhancement of transformation frequency by co-culture medium1898. % Co- Total # Embryos with # % % culture Embryos EmbryogenicEvents Transferred Transferred % Medium Tested Response Created toPhytatrays to Soil TF 1233 660 53.0 107 16.2 11.7 10.6% 1898 628 79.3263 43.5 31.5 30.9%

TABLE 9 Media compositions used in the present invention. Media 1233,1278, 1073, 1071, 1084 are from Cai et al.; U.S. Pat. application Pub.No. 2004/00244075. Medium 1898 is from U.S. Pat. application Pub. No.2008/0124727. 1278 1073 1071 Media 1233 1898 (MSW (MS/6BA) (MSOD)Components/L (co- (co- 50 + BAP) (1^(st) (2^(nd) 1084 (Suppliers)culture) culture) (selection) regeneration) regeneration) (rooting) MSBasal Salts 2.165 g 4.33 g 4.33 g 4.33 g 4.33 g 2.165 g (Phytotech) MSVitamins 10 mL 10 mL 10 mL 0 0 0 (100X) (Phytotech) MS Fromm 0 0 0 1 mL1 mL 0 Vitamins (1000X)* BAP (Sigma) 0 0 0.01 mg 3.5 mg 0 0 Thiamine HCL0.5 mg 0.5 mg 0.5 mg 0 0 0 (Sigma) 2,4-D 3 mg 0.5 mg 0.5 mg 0 0 0(Phytotech) NAA (Sigma) 0 0 0 0 0 0.5 mg IBA (Sigma) 0 0 0 0 0 0.75 mgSucrose 20 g 30 g 30 g 30 g 0 20 g (Phytotech) Glucose 10 g 0 0 0 10 g 0(Phytotech) Maltose 0 0 0 0 20 g 0 (Phytotech) Proline 115 mg 1.38 g1.38 g 1.38 g 0 0 (Sigma) Casamino 0 0.5 g 0.5 g 0.05 g   0.5 0 Acids(Difco) Asparagine 0 0 0 0   0.15 0 monohydrate (Sigma) Myo-inositol 0 00 0 0.1 g 0 (Sigma) Low EEO 5.5 g 5.5 g 0 0 0 0 Agarose (Sigma) Phytagel0 0 3 g 3 g 3 g 3 g (Sigma) Acetosyringone 200 uM 200 uM 0 0 0 0(Aldrich) Carbenicillin 0 50 mg 500 mg 250 mg 250 mg 0 (Phytotech)Glyphosate 0 0 0.1 mM 0.1 mM 0.1 mM 0.1 mM (Gateway Chemical) SilverNitrate 3.4 mg 3.4 mg 3.4 mg 0 0 0 (Sigma) pH   5.2   5.8   5.8   5.8  5.8   5.8 *Comprising 1250 mg/L nicotinic acid (Sigma), 250 mg/Lpyridoxine HCl (Sigma), 250 mg/L thiamine HCl (Sigma), and 250 mg/Lcalcium pantothenate (Sigma).

All of the materials and methods disclosed and claimed herein can bemade and used, as instructed by the above disclosure, and without undueexperimentation, by a person of ordinary skill in the art. Although thematerials and methods of this invention have been described in terms ofpreferred embodiments and illustrative examples, it will be apparent tothose of skill in the art that variations may be applied to thematerials and methods described herein without departing from theconcept, spirit, and scope of the invention. All such similarsubstitutes and modifications apparent to those skilled in the art aredeemed to be within the concept, spirit, and scope of the invention asfurther defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,550,318-   U.S. Pat. No. 5,780,708-   U.S. Pat. No. 6,194,636-   U.S. Pat. No. 6,232,526-   U.S. Pat. No. 7,002,058-   U.S. Pat. No. 7,150,993-   U.S. patent application Ser. No. 10/710,067-   U.S. patent application Ser. No. 11/613,031-   U.S. patent application Ser. No. 12/045,502-   U.S. Patent Publn. 2003/0024014-   U.S. Patent Publn. 2004/0016030-   U.S. Patent Publn. 2004/0126845-   U.S. Patent Publn. 2004/0210958-   U.S. Patent Publn. 2004/0216189-   U.S. Patent Publn. 2004/0244075-   U.S. Patent Publn. 2005/0246786-   U.S. Patent Publn. 2008/0124727-   U.S. Prov. Patent Appln. 60/894,096-   U.S. Prov. Patent Appln. 60/915,066

1-56. (canceled)
 57. A method for preparation of plant embryos suitablefor tissue culture and/or genetic transformation, wherein an apparatusfor holding a kernel or other tissue is centrifuged in a container toapply force to the kernel or other tissue.
 58. An apparatus forobtaining plant embryos suitable for tissue culture and/or genetictransformation comprising (a) at least a first fluid jet for directing amedium onto a corn kernel or other tissue comprising a plant embryo; and(b) an apparatus for holding the kernel or other tissue in the path ofthe medium.
 59. The apparatus of claim 58, wherein the embryo iscomprised within a corn kernel.
 60. The apparatus of claim 59, whereinthe corn kernel is comprised on an ear of corn.
 61. The apparatus ofclaim 58, wherein the apparatus is further defined as comprising a firstand a second fluid jet.
 62. The apparatus of claim 60, wherein theapparatus for holding the kernel comprises means for moving the ear ofcorn relative to the first and second fluid streams to control the angleof contact between the first and second fluid streams and the kernel.63. The apparatus of claim 58, further comprising a detector to identifyexcised endosperm tissue and embryos.
 64. The apparatus of claim 58,further comprising at least a first separator to isolate embryos fromnon-embryo tissue.
 65. The apparatus of claim 58, wherein the apparatusfor holding the kernel or other tissue comprises a sheet or acylindrical sheet?.
 66. The apparatus of claim 58, wherein the apparatusfor holding the kernel or other tissue comprises a mesh, a plurality ofslots; or a pressure cam or screw that applies force to the tissue. 67.The apparatus of claim 58, wherein seed or fruit tissue is held onto theapparatus for holding the kernel or other tissue by a mechanical force,friction, centrifugal force, or a suction force.
 68. The apparatus ofclaim 58, wherein the apparatus for holding the kernel or other tissueis suspended in a gaseous phase, a liquid phase, or is partiallysuspended in gaseous and liquid phases.
 69. The apparatus of claim 58,wherein the apparatus for holding the kernel or other tissue is fixedrelative to a fluid force.
 70. The apparatus of claim 58, wherein theapparatus for holding the kernel or other tissue is movable relative toa fluid force.
 71. The apparatus of claim 64, wherein the separatorseparates embryos suitable for tissue culture from non-embryo tissue bya method selected from the group consisting of size exclusion,differential density and differential hydrophobicity.
 72. The apparatusof claim 71, further defined as comprising a sieve for separating embryofrom non-embryo tissue based on size.
 73. The apparatus of claim 60,wherein the apparatus for holding the kernel comprises at least a firstmotor for moving the ear of corn relative to the fluid jet.
 74. Themethod of claim 57, wherein the kernel is comprised on an ear of corn.75. The method of claim 74, wherein the ear of corn is rotated about itslongitudinal axis.
 76. The method of claim 57, wherein the kernel orother tissue is held onto the apparatus for holding the kernel or othertissue by a mechanical force, a friction force, a centrifugal force, ora suction force.
 77. The method of claim 57, wherein the apparatus forholding a kernel or other tissue comprises a sheet or a cylinder. 78.The method of claim 77, wherein the apparatus for holding a kernel orother tissue comprises plastic or metal.
 79. The method of claim 77,wherein the apparatus for holding a kernel or other tissue comprisesholes or slots.
 80. The method of claim 57, wherein the plant embryo isa maize embryo, a millet embryo, a soybean embryo, a canola embryo, or acotton embryo.
 81. The method of claim 57, wherein the containercomprises multiple plant embryos.
 82. The method of claim 57, whereinthe force is centrifugal force.
 83. The method of claim 57, wherein theplant embryo is separated from other tissue by differential density. 84.The method of claim 57 wherein the top of the kernel is removed prior toapplying the force.
 85. The method of claim 57 wherein the plant embryois ejected from the kernel.